Vegf/dll4 binding agents and uses thereof

ABSTRACT

The present invention relates to VEGF-binding agents, DLL4-binding agents, VEGF/DLL4 bispecific binding agents, and methods of using the agents for treating diseases such as cancer. The present invention provides antibodies that specifically bind human VEGF, antibodies that specifically bind human DLL4, and bispecific antibodies that specifically bind human VEGF and/or human DLL4. The present invention further provides methods of using the agents to inhibit tumor growth. Also described are methods of treating cancer comprising administering a therapeutically effect amount of an agent or antibody of the present invention to a patient having a tumor or cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/928,452, filed Jul. 14, 2020, now allowed, which is a continuation ofU.S. application Ser. No. 15/847,335, filed Dec. 19, 2017, now U.S. Pat.No. 10,730,940, which is a divisional of U.S. application Ser. No.15/401,543, filed Jan. 9, 2017, now U.S. Pat. No. 9,879,084, which is adivisional of U.S. application Ser. No. 15/163,301, filed May 24, 2016,now U.S. Pat. No. 9,574,009, which is a divisional of U.S. applicationSer. No. 14/476,582, filed Sep. 3, 2014, now U.S. Pat. No. 9,376,488,which is a divisional of U.S. application Ser. No. 13/625,417, filedSep. 24, 2012, now U.S. Pat. No. 8,858,941, which claims the benefit ofU.S. Provisional Application No. 61/538,454, filed Sep. 23, 2011, U.S.Provisional Application No. 61/597,409, filed Feb. 10, 2012, and U.S.Provisional Application No. 61/692,978, filed Aug. 24, 2012, each ofwhich is hereby incorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:4488_084000A_Seqlisting_ST26, Size: 125,195 bytes; and Date of Creation:Oct. 28, 2022) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to antibodies and other agentsthat bind VEGF, DLL4, or both VEGF and DLL4, particularlyanti-VEGF/anti-DLL4 bispecific antibodies, as well as to methods ofusing the antibodies or other agents for the treatment of diseases suchas cancer.

BACKGROUND OF THE INVENTION

Angiogenesis plays an important role in the pathogenesis of a number ofdisorders, including solid tumors and metastasis. The production of newblood vessels is essential for providing oxygen and nutrients for thegrowth and spread of a tumor, and therefore angiogenesis is a goodtarget for cancer therapeutics.

Angiogenesis involves a family of proteins acting as angiogenicactivators, including vascular endothelial growth factor (VEGF-A),VEGF-B, VEGF-C, VEGF-E, and their respective receptors (VEGFR-1,VEGFR-2, and VEGFR-3). VEGF-A, also referred to as VEGF or vascularpermeability factor (VPF), exists in several isoforms that arise fromalternative splicing of mRNA of a single VEGF gene, with VEGF₁₆₅ beingthe most biologically relevant isoform.

Anti-VEGF antibodies have been shown to suppress the growth of tumorcells in vitro and in vivo. A humanized anti-VEGF monoclonal antibody,bevacizumab (AVASTIN) has been developed and approved in the UnitedStates as a cancer therapeutic.

The Notch signaling pathway is a universally conserved signaltransduction system. It is involved in cell fate determination duringdevelopment including embryonic pattern formation and post-embryonictissue maintenance. In addition, Notch signaling has been identified asa critical factor in the maintenance of hematopoietic stem cells.

The Notch pathway has been linked to the pathogenesis of bothhematologic and solid tumors and cancers. Numerous cellular functionsand microenvironmental cues associated with tumorigenesis have beenshown to be modulated by Notch pathway signaling, including cellproliferation, apoptosis, adhesion, and angiogenesis (Leong et al.,2006, Blood, 107:2223-2233). In addition, Notch receptors and/or Notchligands have been shown to play potential oncogenic roles in a number ofhuman cancers, including acute myelogenous leukemia, B cell chroniclymphocytic leukemia, Hodgkin lymphoma, multiple myeloma, T-cell acutelymphoblastic leukemia, brain cancer, breast cancer, cervical cancer,colon cancer, lung cancer, pancreatic cancer, prostate cancer, and skincancer. (Leong et al., 2006, Blood, 107:2223-2233).

Delta-like 4 ligand (DLL4) is an important component of the Notchpathway and has been identified as a target for cancer therapy. DLL4 isa Notch ligand, characterized by an N-terminal domain, aDelta/Serrate/Lag-2 (DSL) domain and tandem EGF-like repeats within theextracellular domain. It has been reported that DLL4 is induced by VEGFand that DLL4 may act as a negative feedback regulator for vascularproliferation.

Anti-DLL4 antibodies have been shown to enhance angiogenic sprouting andbranching which leads to non-productive angiogenesis and decreased tumorgrowth (Noguera-Troise et al., 2006, Nature, 444:1032-1037). Inaddition, an anti-DLL4 antibody, 21M18, has been shown to inhibit tumorgrowth and reduce the frequency of cancer stem cells in xenograft tumormodels (Hoey et al., 2009, Cell Stem Cell, 5:168-177; U.S. Pat. No.7,750,124).

Although there have been significant strides in development ofmonoclonal antibodies for use in cancer treatments, there is still greatpotential for further improvements. One class of antibody molecules withthe promise of enhanced potency and/or reduced side effects (e.g.,toxicity) is bispecific antibodies.

Early bispecific molecules were mainly generated using chemicalcross-linking of two antibodies, or were hybrid hybridomas or“quadromas”. One success of the quadroma format is triomabs, which aremouse/rat combinations that demonstrate a preferential species-specificheavy/light chain pairing. More recently, advances in antibodyengineering have provided a wide variety of new antibody formats,including, but not limited to, tandem scFv (bi-scFv), diabodies, tandemdiabodies (tetra-bodies), single chain diabodies, and dual variabledomain antibodies.

It is one of the objectives of the present invention to provide improvedmolecules for cancer treatment, particularly bispecific antibodies thatspecifically bind human VEGF and human DLL4.

SUMMARY OF THE INVENTION

The present invention provides binding agents, such as antibodies, thatbind VEGF, DLL4, or both VEGF and DLL4 (VEGF/DLL4-binding agents), aswell as compositions, such as pharmaceutical compositions, comprisingthe binding agents. Binding agents that bind VEGF or DLL4, as well as atleast one additional antigen or target, and pharmaceutical compositionsof such binding agents, are also provided. In certain embodiments, thebinding agents are novel polypeptides, such as antibodies, antibodyfragments, and other polypeptides related to such antibodies. In certainembodiments, the binding agents are antibodies that specifically bindhuman VEGF. In some embodiments, the binding agents are antibodies thatspecifically bind human DLL4. In some embodiments, the binding agentsare bispecific antibodies that specifically bind human VEGF and humanDLL4. The invention further provides methods of inhibiting the growth ofa tumor by administering the binding agents to a subject with a tumor.The invention further provides methods of treating cancer byadministering the binding agents to a subject in need thereof. In someembodiments, the methods of treating cancer or inhibiting tumor growthcomprise targeting cancer stem cells with the binding agents. In certainembodiments, the methods comprise reducing the frequency of cancer stemcells in a tumor, reducing the number of cancer stem cells in a tumor,reducing the tumorigenicity of a tumor, and/or reducing thetumorigenicity of a tumor by reducing the number or frequency of cancerstem cells in the tumor.

In one aspect, the invention provides a binding agent, such as anantibody, that specifically binds human VEGF. In some embodiments, thebinding agent inhibits binding of VEGF to at least one VEGF receptor. Insome embodiments, the binding agent inhibits binding of VEGF to VEGFR-1and/or VEGFR-2. In some embodiments, the binding agent modulatesangiogenesis. In certain embodiments, the antibody or other bindingagent further specifically binds to and/or inhibits human DLL4 inaddition to human VEGF.

In some embodiments, the binding agent is an antibody which comprises aheavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19); and a light chain CDR1comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprisingAASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG(SEQ ID NO:22).

In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region having at least 80% sequence identity toSEQ ID NO:11; and/or a light chain variable region having at least 80%sequence identity to SEQ ID NO:12. In certain embodiments, the bindingagent comprises a heavy chain variable region having at least 90%sequence identity to SEQ ID NO:11; and/or a light chain variable regionhaving at least 90% sequence identity to SEQ ID NO:12. In certainembodiments, the binding agent comprises a heavy chain variable regionhaving at least 95% sequence identity to SEQ ID NO:11; and/or a lightchain variable region having at least 95% sequence identity to SEQ IDNO:12. In certain embodiments, the binding agent is an antibody thatcomprises a heavy chain variable region of SEQ ID NO:11; and/or a lightchain variable region of SEQ ID NO:12.

In some embodiments, the binding agent is antibody 219R45,219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, or 219R45-MB-21R83.

In another aspect, the invention provides a binding agent, such as anantibody, that specifically binds human DLL4. In some embodiments, thebinding agent inhibits binding of DLL4 to at least one Notch receptor.In some embodiments, the binding agent inhibits binding of DLL4 toNotch1, Notch2, Notch3, and/or Notch4. In some embodiments, the bindingagent inhibits Notch signaling. In some embodiments, the binding agentpromotes unproductive angiogenesis. In certain embodiments, the antibodyor other binding agent further specifically binds to and/or inhibitshuman VEGF in addition to human DLL4.

In some embodiments, the binding agent is an antibody that binds humanDLL4 and comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13)or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprisingYIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁ is serine or alanine,X₂ is serine, asparagine, or glycine, X₃ is asparagine or lysine, and X₄is glysine, arginine, or aspartic acid, and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the antibody comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chainCDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).

In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region having at least 90% or at least 95%sequence identity to SEQ ID NO:10; and/or a light chain variable regionhaving at least 90% or at least 95% sequence identity to SEQ ID NO:12.In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region of SEQ ID NO:10; and a light chainvariable region of SEQ ID NO:12.

In some embodiments, the binding agent is antibody 21R79 or antibody219R45-MB-21R79.

In some embodiments, the binding agent is an antibody which comprises aheavy chain CDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ IDNO:79), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region having at least 90% or at least 95%sequence identity to SEQ ID NO:58; and/or a light chain variable regionhaving at least 90% or at least 95% sequence identity to SEQ ID NO:12.In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region of SEQ ID NO:58; and a light chainvariable region of SEQ ID NO:12.

In some embodiments, the binding agent is antibody 21R75 or antibody219R45-MB-21R75.

In some embodiments, the binding agent is an antibody which comprises aheavy chain CDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ IDNO:79), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region having at least 90% or at least 95%sequence identity to SEQ ID NO:64; and/or a light chain variable regionhaving at least 90% or at least 95% sequence identity to SEQ ID NO:12.In certain embodiments, the binding agent is an antibody that comprisesa heavy chain variable region of SEQ ID NO:64; and a light chainvariable region of SEQ ID NO:12.

In some embodiments, the binding agent is antibody 21R83 or antibody219R45-MB-21R83.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the binding agent is a bispecific antibody. In someembodiments, the bispecific antibody specifically binds human VEGF and asecond target. In some embodiments, the bispecific antibody specificallybinds human DLL4 and a second target. In some embodiments, thebispecific antibody specifically binds both human VEGF and human DLL4.In some embodiments, the bispecific antibody modulates angiogenesis. Incertain embodiments, the bispecific antibody inhibits Notch signaling.In some embodiments, the bispecific antibody modulates angiogenesis andinhibits Notch signaling. In some embodiments, the bispecific antibodyreduces the number of frequency of cancer stem cells. In certainembodiments, the bispecific antibody comprises two identical lightchains. In certain embodiments the bispecific antibody is an IgGantibody (e.g., IgG2).

In some embodiments, the bispecific antibody comprises: a firstantigen-binding site that specifically binds human VEGF, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19).In some embodiments, the bispecific antibody further comprises: a lightchain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecificantibody comprises: a first antigen-binding site that specifically bindshuman VEGF, wherein the first antigen-binding site comprises (a) a heavychain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19), and (b) a light chain CDR1comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprisingAASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG(SEQ ID NO:22).

In certain embodiments, the bispecific antibody comprises: a firstantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprisingTAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2comprising YIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁ is serine oralanine, X₂ is serine, asparagine, or glycine, X₃ is asparagine orlysine, and X₄ is glysine, arginine, or aspartic acid, and a heavy chainCDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDR1comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprisingAASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG(SEQ ID NO:22). In some embodiments, the bispecific antibody comprises:a first antigen-binding site that specifically binds human DLL4, whereinthe first antigen-binding site comprises a heavy chain CDR1 comprisingTAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG(SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQID NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:16). In some embodiments, thebispecific antibody further comprises: a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the the bispecific antibody comprises: afirst antigen-binding site that specifically binds human DLL4, whereinthe first antigen-binding site comprises (a) a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIANYNRATNYNQKFKG (SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15),YIAGYKDATNYNQKFKG (SEQ ID NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In some embodiments, the bispecific antibody comprises: a) a firstantigen-binding site that specifically binds human VEGF, and b) a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19);wherein the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chainCDR2 comprising YIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁ isserine or alanine, X₂ is serine, asparagine, or glycine, X₃ isasparagine or lysine, and X₄ is glysine, arginine, or aspartic acid, anda heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a lightchain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the bispecificantibody comprises: a) a first antigen-binding site that specificallybinds human VEGF, and b) a second antigen-binding site that specificallybinds human DLL4, wherein the first antigen-binding site comprises aheavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the secondantigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ IDNO:14), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16);and wherein both the first and second antigen-binding sites comprise alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, thebispecific antibody comprises: a) a first antigen-binding site thatspecifically binds human VEGF, and b) a second antigen-binding site thatspecifically binds human DLL4, wherein the first antigen-binding sitecomprises a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavychain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavychain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the secondantigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ IDNO:15), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16);and wherein both the first and second antigen-binding sites comprise alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the abispecific antibody comprises: a) a first antigen-binding site thatspecifically binds human VEGF, and b) a second antigen-binding site thatspecifically binds human DLL4, wherein the first antigen-binding sitecomprises a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavychain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavychain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the secondantigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ IDNO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16);and wherein both the first and second antigen-binding sites comprise alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, thebispecific antibody comprises: a) a first antigen-binding site thatspecifically binds human VEGF, and b) a second antigen-binding site thatspecifically binds human DLL4, wherein the first antigen-binding sitecomprises a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavychain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavychain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the secondantigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ IDNO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16);and wherein both the first and second antigen-binding sites comprise alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In some embodiments, the bispecific antibody that specifically bindshuman VEGF, and comprises: a heavy chain variable region having at least90% sequence identity to SEQ ID NO:11, and/or a light chain variableregion having at least 90% sequence identity to SEQ ID NO:12. In someembodiments, the bispecific antibody specifically binds human VEGF, andcomprises: a heavy chain variable region having at least 95% sequenceidentity to SEQ ID NO:11, and/or a light chain variable region having atleast 95% sequence identity to SEQ ID NO:12.

In some embodiments, the bispecific antibody specifically binds humanDLL4, and comprises: a heavy chain variable region having at least 90%sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ IDNO:64; and/or a light chain variable region having at least 90% sequenceidentity to SEQ ID NO:12. In some embodiments, the bispecific antibodyspecifically binds human DLL4, and comprises: a heavy chain variableregion having at least 95% sequence identity to SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:58, or SEQ ID NO:64; and/or a light chain variableregion having at least 95% sequence identity to SEQ ID NO:12.

In some embodiments, the bispecific antibody specifically binds humanVEGF and human DLL4, and comprises: (a) a first heavy chain variableregion having at least 90% sequence identity to SEQ ID NO:11; (b) asecond heavy chain variable region having at least 90% sequence identityto SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64; and (c) afirst and a second light chain variable region having at least 90%sequence identity to SEQ ID NO:12. In some embodiments, the VEGF/DLL4bispecific antibody comprises (a) a first heavy chain variable regionhaving at least 95% sequence identity to SEQ ID NO:11; (b) a secondheavy chain variable region having at least 95% sequence identity to SEQID NO:9; and (c) a first and a second light chain variable region havingat least 95% sequence identity to SEQ ID NO:12. In some embodiments, theVEGF/DLL4 bispecific antibody comprises (a) a first heavy chain variableregion having at least 95% sequence identity to SEQ ID NO:11; (b) asecond heavy chain variable region having at least 95% sequence identityto SEQ ID NO:10; and (c) a first and a second light chain variableregion having at least 95% sequence identity to SEQ ID NO:12. In someembodiments, the VEGF/DLL4 bispecific antibody comprises (a) a firstheavy chain variable region having at least 95% sequence identity to SEQID NO:11; (b) a second heavy chain variable region having at least 95%sequence identity to SEQ ID NO:58; and (c) a first and a second lightchain variable region having at least 95% sequence identity to SEQ IDNO:12. In some embodiments, the VEGF/DLL4 bispecific antibody comprises(a) a first heavy chain variable region having at least 95% sequenceidentity to SEQ ID NO:11; (b) a second heavy chain variable regionhaving at least 95% sequence identity to SEQ ID NO:64; and (c) a firstand a second light chain variable region having at least 95% sequenceidentity to SEQ ID NO:12.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising (a) a first antigen-binding site that binds humanVEGF with a K_(D) between about 0.1 nM and about 1.0 nM and (b) a secondantigen-binding site that specifically binds human DLL4 with a K_(D)between about 0.1 nM and about 20 nM. In certain embodiments, thebispecific antibody comprises two identical light chains.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody selected from the group consisting of 219R45-MB-21M18,219R45-MB-21R79, 219R45-MB-21R75, and 219R45-MB-21R83.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and/or embodiments described elsewhere herein, the bindingagent or antibody is isolated.

In another aspect, the invention provides a polypeptide selected fromthe group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some embodiments, thepolypeptide is isolated. In certain embodiments, the polypeptide issubstantially pure. In certain embodiments, the polypeptide is anantibody or part of an antibody, such as an antibody fragment.

In another aspect, the invention provides isolated polynucleotidemolecules comprising a polynucleotide that encodes the binding agentsand/or polypeptides of each of the aforementioned aspects, as well asother aspects and/or embodiments described herein. In some embodiments,the polynucleotide comprises a sequence selected from the groupconsisting of: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:50, SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ IDNO:74. The invention further provides expression vectors that comprisethe polynucleotides, as well as cells that comprise the expressionvectors and/or the polynucleotides. In some embodiments, the cell is aprokaryotic cell or a eukaryotic cell.

In other aspects, the invention provides methods of inhibiting growth ofa tumor, comprising contacting the tumor with an effective amount of anantibody (or other binding agent) that binds VEGF, DLL4, or both VEGFand DLL4, including each of those antibodies (or other binding agents)described herein.

In another aspect, the invention provides a method of inhibiting thegrowth of a tumor in a subject, comprising administering to the subjecta therapeutically effective amount of an antibody (or other bindingagent) that binds VEGF, DLL4, or both VEGF and DLL4, including each ofthose antibodies (or other binding agents) described herein.

In another aspect, the invention provides a method of modulatingangiogenesis in a subject, comprising administering to the subject atherapeutically effective amount of an antibody (or other binding agent)that binds VEGF, DLL4, or both VEGF and DLL4, including each of thoseantibodies (or other binding agents) described herein.

In another aspect, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering to thesubject a therapeutically effective amount of an antibody (or otherbinding agent) that binds VEGF, DLL4, or both VEGF and DLL4, includingeach of those antibodies (or other binding agents) described herein.

In another aspect, the invention provides a method of reducing thetumorigenicity of a tumor in a subject by reducing the frequency ofcancer stem cells in the tumor, comprising administering to the subjecta therapeutically effective amount of an antibody (or other bindingagent) that binds VEGF, DLL4, or both VEGF and DLL4, including each ofthose antibodies (or other binding agents) described herein.

In other aspects, the invention provides methods of treating cancer in asubject, comprising administering to the subject a therapeuticallyeffective amount of an antibody (or other binding agent) that bindsVEGF, DLL4, or both VEGF and DLL4, including each of those antibodies(or other binding agents) described herein.

Pharmaceutical compositions comprising a binding agent (e.g., antibody)described herein and a pharmaceutically acceptable carrier are furtherprovided, as are cell lines that express and/or produce the bindingagents. Methods of treating cancer and/or inhibiting tumor growth in asubject (e.g., a human) comprising administering to the subject aneffective amount of a composition comprising the binding agents are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A, 1B, and 1C. FIG. 1A) Heavy chain and light chain CDRs ofanti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18,219R45-MB-21R79, 219R45-MB-21R75, and 219R45-MB-21R83; FIG. 1B) Heavychain and light chain variable region SEQ ID NOs; FIG. 1C) Heavy chainand light chain SEQ ID NOs.

FIG. 2 . HTRF assay for simultaneous binding of bispecific antibodies tohuman VEGF and human DLL4. Results are reported in Relative FluorescenceUnits (RFU), which represent the ratio of the relative fluorescenceintensity at 665 nm to the relative fluorescence intensity at 620 nm.219R45-MB-21M18 (-●-); 219R45-MB-21R79 (-▪-); 219R45 plus 21M18 (-▴-);219R45 plus 21R79 (-□-); 219R45 (-▾-); 21M18 (—⋄—); 21R79 (-∘-); controlantibody LZ-1 (-Δ-).

FIG. 3 . Inhibition of VEGF-induced HUVEC proliferation byanti-VEGF/anti-DLL4 bispecific antibodies. Fluorescence intensity isread using an excitation wavelength of 530 nm and an emission wavelengthof 590. 219R45-MB-21M18 (-●-); 219R45-MB-21R79 (-▴-); 219R45 (-▪-);Medium with no VEGF (—⋄—).

FIG. 4 . Inhibition of DLL4-induced Notch signaling byanti-VEGF/anti-DLL4 bispecific antibodies. Luciferase activity wasmeasured using a dual luciferase assay kit with firefly luciferaseactivity normalized to Renilla luciferase activity. 219R45-MB-21M18(-●-); 219R45-MB-21R79 (-▪-); 21M18 (-∘-); 21R79 (-□-).

FIG. 5 . Inhibition of colon tumor growth in vivo by ananti-VEGF/anti-DLL4 bispecific antibody. OMP-C8 colon tumor cells wereinjected subcutaneously into a human skin graft in NOD/SCID mice. Micewere treated with control antibody (-▪-), anti-hDLL4 antibody 21M18(-▴-), anti-VEGF antibody bevacizumab (-∘-), or anti-VEGF/anti-DLL4bispecific antibody 219R45-MB-21M18 (-▾-). Data is shown as tumor volume(photons/sec) over days post-treatment. Antibodies were administeredintraperitoneally at a dose of 25 mg/kg once a week.

FIG. 6 . Tumorigenicity of pancreatic tumor cells after treatment withanti-VEGF/anti-DLL4 bispecific antibodies. OMP-PN8 tumor cells from micetreated with control antibody, anti-hDLL4 antibody 21M18, anti-VEGFantibody bevacizumab, or anti-VEGF/anti-DLL4 bispecific antibodies219R45-MB-21M18 or 219R45-MB-21R79 with or without gemcitabine wereprocessed to single cell suspensions, and serially transplanted intomice. 90 cells from each treatment group were injected subcutaneouslyinto NOD/SCID mice. Tumors were allowed to grow with no treatment. Datais shown as tumor volume (mm³) on day 55. Tumor frequency is shown asnumber of tumors over total number of mice injected in each group.

FIG. 7 . Bispecific antibody ELISA. Bispecific antibodies219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, and 219R45-MB-21R83were diluted in blocking buffer (lx PBS, 0.1% gelatin, 0.1%Polysorbate-20, pH 7.4) containing 2 μg/ml biotin-DLL4-hFc. Theantibodies were serially diluted 3-fold from 500 ng/ml to 0.008 ng/ml.The antibody samples were incubated for 2 hours in blocking buffercontaining the biotin-DLL4-hFc. After incubation, the antibody sampleswere transferred to a VEGF-coated assay plate (100 ul/well) andincubated for 2 hours. Streptavidin-HRP was added to each well andincubated for 1 hr. TMB substrate was added to the wells with a 10minute color development and the reaction was stopped with 2M sulfuricacid. Absorbance was read at 450-650 nm and the data analyzed using the4-parameter fit within the Softmax Pro analysis program.

FIG. 8 . Imaged capillary isoelectric focusing of anti-VEGF/anti-DLL4bispecific antibodies.

FIG. 9 . Inhibition of colon tumor growth by anti-VEGF/anti-DLL4bispecific antibodies in tumor recurrence model. OMP-C8 colon tumorcells were injected subcutaneously in NOD/SCID mice. Mice were treatedwith control antibody (-▪-), anti-hDLL4 antibody 21M18 (-●-), anti-VEGFantibody bevacizumab (-▴-), a combination of 21M18 and bevacizumabanti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21M18 (—▾—), oranti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R79 (-∘-), all incombination with irinotecan. Antibodies 21M18 and bevacizumab wereadministered intraperitoneally at a dose of 7.5 mg/kg once a week,bispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79 wereadministered intraperitoneally at a dose of 15 mg/kg once a week, andirinotecan was administered for the first 4 weeks at a dose of 45 mg/kg.Data are shown as tumor volume (mm³) over days post-treatment.

FIG. 10 . Tumorigenicity of OMP-C3 colon tumor cells after treatmentwith anti-VEGF/anti-DLL4 bispecific antibodies. Tumors from mice treatedwith control antibody, anti-hDLL4 antibody 21M18, anti-VEGF antibodybevacizumab, a combination of 21M18 and bevacizumab, oranti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18 or219R45-MB-21R79 with or without irinotecan were processed to single cellsuspensions, and serially transplanted into mice. 150 cells from eachtreatment group were injected subcutaneously into NOD/SCID mice. Tumorswere allowed to grow with no treatment. Data are shown as tumor volume(mm³) on day 68.

FIGS. 11A and 11B. Inhibition of colon tumor growth in vivo byanti-VEGF/anti-DLL4 bispecific antibodies. OMP-C8 colon tumor cells wereinjected subcutaneously into NOD/SCID mice. Mice were treated withcontrol antibody (-▪-), anti-VEGF antibody bevacizumab (-▴-), oranti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18 (—⋄—),219R45-MB-21R75 (-●-), 219R45-MB-21R79 (-∘-), or 219R45-MB-21R83 (-▾-).Mice were treated with antibodies as single agents (FIG. 11A) or incombination with irinotecan (FIG. 11B). Antibodies were administeredintraperitoneally at a dose of 15 mg/kg once a week and irinotecan at adose of 7.5 mg/kg one a week. Data are shown as tumor volume (mm³) overdays post-treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel binding agents, including but notlimited to polypeptides such as antibodies, that bind VEGF and/or DLL4(e.g., a VEGF/DLL4 binding agent). Related polypeptides andpolynucleotides, compositions comprising the VEGF/DLL4-binding agents,and methods of making the VEGF/DLL4-binding agents are also provided.Methods of using the novel VEGF/DLL4-binding agents, such as methods ofinhibiting tumor growth, methods of treating cancer, methods of reducingtumorigenicity of a tumor, methods of reducing the frequency of cancerstem cells in a tumor, and/or methods of modulating angiogenesis, arefurther provided.

A monoclonal antibody that specifically binds human VEGF has beenidentified, 219R45. This antibody has a binding affinity for human VEGFof about 0.67 nM, and a binding affinity for mouse VEGF of about 23 nM.Several monoclonal antibodies that specifically bind human DLL4 havebeen identified, 21R79, 21R75 and 21R83. Antibody 21R79 has a bindingaffinity for human DLL4 of less than 0.1 nM. Bispecific antibodies thatspecifically bind human VEGF and human DLL4 have been produced,219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, and 219R45-MB-21R83(CDR sequences in FIG. 1 ). As used herein, the “MB” within an antibodyname refers to “monovalent/bispecific”. Bispecific antibody219R45-MB-21M18 has a binding affinity for human VEGF of less than 1.0nM and a binding affinity for human DLL4 of about 16 nM. Bispecificantibody 219R45-MB-21R79 has a binding affinity for human VEGF of lessthan 1.0 nM and a binding affinity for human DLL4 of less than 1.0 nM.Bispecific antibody 219R45-MB-21R75 has a binding affinity for humanDLL4 of about 5 nM, while bispecific antibody 219R45-MB-21R83 has abinding affinity for human DLL4 of about 1 nM. Bispecific antibodies219R45-MB-21M18 and 219R45-MB-21R79 bind mouse VEGF (Example 1, Table3). Anti-VEGF/anti-DLL4 bispecific antibodies bind human VEGF and humanDLL4 simultaneously (Example 2, FIG. 2 ). Anti-VEGF/anti-DLL4 bispecificantibodies inhibit VEGF-induced proliferation of HUVEC cells (Example 3,FIG. 3 ). Anti-VEGF/anti-DLL4 bispecific antibodies inhibit DLL4-inducedNotch signaling (Example 4, FIG. 4 ). Anti-VEGF/anti-DLL4 bispecificantibodies inhibit tumor growth (Examples 5, 9, 11 and FIGS. 5, 9, 11 ).Anti-VEGF/anti-DLL4 bispecific antibodies inhibit tumorigenicity(Examples 6 and 10 and FIGS. 6, 10 ). Anti-VEGF/anti-DLL4 bispecificantibodies bind both VEGF and DLL4 in a bispecific ELISA (Example 7,FIG. 7 ). Anti-VEGF/anti-DLL4 bispecific antibodies are isolated andpurified to a product comprising at least 90% heterodimeric antibody(Example 8, Table 7).

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “antibody” as used herein refers to an immunoglobulin moleculethat recognizes and specifically binds a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing, through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term encompasses intact polyclonal antibodies, intactmonoclonal antibodies, single chain antibodies, antibody fragments (suchas Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv)antibodies, multispecific antibodies such as bispecific antibodies,monospecific antibodies, monovalent antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins comprising anantigen-binding site of an antibody, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site (i.e.,antigen-binding site) as long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on theidentity of their heavy chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well-known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules, including but not limited to, toxins andradioisotopes.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. “Antibody fragment” as used herein comprises anantigen-binding site or epitope-binding site.

The term “variable region” of an antibody refers to the variable regionof an antibody light chain, or the variable region of an antibody heavychain, either alone or in combination. The variable regions of the heavyand light chains each consist of four framework regions (FR) connectedby three complementarity determining regions (CDRs), also known as“hypervariable regions”. The CDRs in each chain are held together inclose proximity by the framework regions and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site ofthe antibody. There are at least two techniques for determining CDRs:(1) an approach based on cross-species sequence variability (i.e., Kabatet al., 1991, Sequences of Proteins of Immunological Interest, 5thEdition, National Institutes of Health, Bethesda, Md.), and (2) anapproach based on crystallographic studies of antigen-antibody complexes(Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition,combinations of these two approaches are sometimes used in the art todetermine CDRs.

The term “monoclonal antibody” as used herein refers to a homogeneousantibody population involved in the highly specific recognition andbinding of a single antigenic determinant or epitope. This is incontrast to polyclonal antibodies that typically include a mixture ofdifferent antibodies directed against a variety of different antigenicdeterminants. The term “monoclonal antibody” encompasses both intact andfull-length monoclonal antibodies as well as antibody fragments (e.g.,Fab, Fab′, F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteinscomprising an antibody portion, and any other modified immunoglobulinmolecule comprising an antigen recognition site (antigen-binding site).Furthermore, “monoclonal antibody” refers to such antibodies made by anynumber of techniques, including but not limited to, hybridomaproduction, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” as used herein refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues of the CDRs are replaced by residuesfrom the CDRs of a non-human species (e.g., mouse, rat, rabbit, orhamster) that have the desired specificity, affinity, and/or bindingcapability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science,239:1534-1536). In some instances, the Fv framework region residues of ahuman immunoglobulin are replaced with the corresponding residues in anantibody from a non-human species that has the desired specificity,affinity, and/or binding capability. The humanized antibody can befurther modified by the substitution of additional residues either inthe Fv framework region and/or within the replaced non-human residues torefine and optimize antibody specificity, affinity, and/or bindingcapability. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two or three, variabledomains containing all or substantially all of the CDRs that correspondto the non-human immunoglobulin whereas all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody can also comprise at least a portion ofan immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. Examples of methods used to generate humanizedantibodies are described in, for example, U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein refers to an antibody producedby a human or an antibody having an amino acid sequence corresponding toan antibody produced by a human. A human antibody may be made using anyof the techniques known in the art. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-human CDRs.

The term “chimeric antibody” as used herein refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. Typically, the variable region of bothlight and heavy chains corresponds to the variable region of antibodiesderived from one species of mammals (e.g., mouse, rat, rabbit, etc.)with the desired specificity, affinity, and/or binding capability, whilethe constant regions correspond to sequences in antibodies derived fromanother species (usually human).

The phrase “affinity-matured antibody” as used herein refers to anantibody with one or more alterations in one or more CDRs thereof thatresult in an improvement in the affinity of the antibody for antigen,compared to a parent antibody that does not possess thosealterations(s). The definition also includes alterations in non-CDRresidues made in conjunction with alterations to CDR residues. Preferredaffinity-matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity-matured antibodies areproduced by procedures known in the art. For example, Marks et al.,1992, Bio/Technology 10:779-783, describes affinity maturation by VH andVL domain shuffling. Random mutagenesis of CDR and/or framework residuesis described by Barbas et al., 1994, PNAS, 91:3809-3813; Schier et al.,1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol., 154:3310-9; and Hawkins et al., 1992,J. Mol. Biol., 226:889-896. Site-directed mutagenesis may also be usedto obtain affinity-matured antibodies.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids (also referredto as linear epitopes) are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding (also referred to asconformational epitopes) are typically lost upon protein denaturing. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation.

The terms “heteromultimeric molecule” or “heteromultimer” or“heteromultimeric complex” or “heteromultimeric polypeptide” are usedinterchangeably herein to refer to a molecule comprising at least afirst polypeptide and a second polypeptide, wherein the secondpolypeptide differs in amino acid sequence from the first polypeptide byat least one amino acid residue. The heteromultimeric molecule cancomprise a “heterodimer” formed by the first and second polypeptide orcan form higher order tertiary structures where additional polypeptidesare present.

The terms “antagonist” and “antagonistic” as used herein refer to anymolecule that partially or fully blocks, inhibits, reduces, orneutralizes a biological activity of a target and/or signaling pathway(e.g., the Notch pathway). The term “antagonist” is used herein toinclude any molecule that partially or fully blocks, inhibits, reduces,or neutralizes the activity of a protein. Suitable antagonist moleculesspecifically include, but are not limited to, antagonist antibodies orantibody fragments.

The terms “modulation” and “modulate” as used herein refer to a changeor an alteration in a biological activity. Modulation includes, but isnot limited to, stimulating or inhibiting an activity. Modulation may bean increase or a decrease in activity (e.g., a decrease in angiogenesisor an increase in angiogenesis), a change in binding characteristics, orany other change in the biological, functional, or immunologicalproperties associated with the activity of a protein, pathway, or otherbiological point of interest.

The terms “selectively binds” or “specifically binds” mean that abinding agent or an antibody reacts or associates more frequently, morerapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein, or target moleculethan with alternative substances, including unrelated proteins. Incertain embodiments “specifically binds” means, for instance, that anantibody binds a protein with a K_(D) of about 0.1 mM or less, but moreusually less than about 1 μM. In certain embodiments, “specificallybinds” means that an antibody binds a target at times with a K_(D) of atleast about 0.1 μM or less, at other times at least about 0.01 μM orless, and at other times at least about 1 nM or less. Because of thesequence identity between homologous proteins in different species,specific binding can include an antibody that recognizes a protein inmore than one species (e.g., human VEGF and mouse VEGF). Likewise,because of homology within certain regions of polypeptide sequences ofdifferent proteins, specific binding can include an antibody (or otherpolypeptide or binding agent) that recognizes more than one protein(e.g., human VEGF-A and human VEGF-B). It is understood that, in certainembodiments, an antibody or binding moiety that specifically binds afirst target may or may not specifically bind a second target. As such,“specific binding” does not necessarily require (although it caninclude) exclusive binding, i.e. binding to a single target. Thus, anantibody may, in certain embodiments, specifically bind more than onetarget. In certain embodiments, multiple targets may be bound by thesame antigen-binding site on the antibody. For example, an antibody may,in certain instances, comprise two identical antigen-binding sites, eachof which specifically binds the same epitope on two or more proteins. Incertain alternative embodiments, an antibody may be multispecific andcomprise at least two antigen-binding sites with differingspecificities. By way of non-limiting example, a bispecific antibody maycomprise one antigen-binding site that recognizes an epitope on oneprotein (e.g., human VEGF) and further comprise a second, differentantigen-binding site that recognizes a different epitope on a secondprotein (e.g., human DLL4). Generally, but not necessarily, reference tobinding means specific binding.

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because the polypeptides of this invention may be basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to polymers of nucleotides of any length, and includeDNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase.

“Conditions of high stringency” may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example15 mM sodium chloride/1.5 mM sodium citrate/0.1% sodium dodecyl sulfateat 50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 in 5×SSC (0.75M NaCl, 75 mM sodium citrate) at 42° C.;or (3) employ during hybridization 50% formamide in 5×SSC, 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 50% formamide, followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known in the art. These include,but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variations thereof. In some embodiments, two nucleic acidsor polypeptides of the invention are substantially identical, meaningthey have at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. In some embodiments, identity existsover a region of the sequences that is at least about 10, at least about20, at least about 40-60 residues, at least about 60-80 residues inlength or any integral value therebetween. In some embodiments, identityexists over a longer region than 60-80 residues, such as at least about80-100 residues, and in some embodiments the sequences are substantiallyidentical over the full length of the sequences being compared, such asthe coding region of a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen to which the polypeptide or antibody binds. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate antigen binding are well-known in the art.

The term “vector” as used herein means a construct, which is capable ofdelivering, and usually expressing, one or more gene(s) or sequence(s)of interest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid, or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells, orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, a polypeptide, antibody, polynucleotide, vector, cell, orcomposition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, blastoma, sarcoma, andhematologic cancers such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass oftissue that results from excessive cell growth or proliferation, eitherbenign (noncancerous) or malignant (cancerous) including pre-cancerouslesions.

The term “metastasis” as used herein refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body with the development of a similar cancerous lesion at a newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumorinitiating cell” are used interchangeably herein and refer to cells froma cancer or tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more types ofdifferentiated cell progeny wherein the differentiated cells havereduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties confer on the cancer stem cells the ability to form orestablish a tumor or cancer upon serial transplantation into animmunocompromised host (e.g., a mouse) compared to the majority of tumorcells that fail to form tumors. Cancer stem cells undergo self-renewalversus differentiation in a chaotic manner to form tumors with abnormalcell types that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total populationof cells derived from a cancer or tumor or pre-cancerous lesion,including both non-tumorigenic cells, which comprise the bulk of thecancer cell population, and tumorigenic stem cells (cancer stem cells).As used herein, the terms “cancer cell” or “tumor cell” will be modifiedby the term “non-tumorigenic” when referring solely to those cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional featuresof a cancer stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of arandom sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised hosts (e.g., mice). Thisdefinition also includes enriched and/or isolated populations of cancerstem cells that form palpable tumors upon serial transplantation intoimmunocompromised hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The term “pharmaceutically acceptable” refers to a product or compoundapproved (or approvable) by a regulatory agency of the Federalgovernment or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, includinghumans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant”or “acceptable pharmaceutical carrier” refer to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone binding agent (e.g., an antibody) of the present disclosure, andwhich does not destroy the activity of the binding agent. The excipient,carrier or adjuvant should be nontoxic when administered with a bindingagent in doses sufficient to deliver a therapeutic effect.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a binding agent, an antibody,polypeptide, polynucleotide, small organic molecule, or other drugeffective to “treat” a disease or disorder in a subject or mammal. Inthe case of cancer, the therapeutically effective amount of a drug(e.g., an antibody) has a therapeutic effect and as such can reduce thenumber of cancer cells; decrease tumorigenicity, tumorigenic frequencyor tumorigenic capacity; reduce the number or frequency of cancer stemcells; reduce the tumor size; reduce the cancer cell population; inhibitand/or stop cancer cell infiltration into peripheral organs including,for example, the spread of cancer into soft tissue and bone; inhibitand/or stop tumor or cancer cell metastasis; inhibit and/or stop tumoror cancer cell growth; relieve to some extent one or more of thesymptoms associated with the cancer; reduce morbidity and mortality;improve quality of life; or a combination of such effects. To the extentthe agent, for example an antibody, prevents growth and/or killsexisting cancer cells, it can be referred to as cytostatic and/orcytotoxic.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. In some embodiments, a subjectis successfully “treated” according to the methods of the presentinvention if the patient shows one or more of the following: a reductionin the number of or complete absence of cancer cells; a reduction in thetumor size; inhibition of or an absence of cancer cell infiltration intoperipheral organs including the spread of cancer cells into soft tissueand bone; inhibition of or an absence of tumor or cancer cellmetastasis; inhibition or an absence of cancer growth; relief of one ormore symptoms associated with the specific cancer; reduced morbidity andmortality; improvement in quality of life; reduction in tumorigenicity;reduction in the number or frequency of cancer stem cells; or somecombination of effects.

As used in the present disclosure and claims, the singular forms “a”,“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.It is also understood that wherever embodiments are described hereinwith the language “consisting essentially of” otherwise analogousembodiments described in terms of “consisting of” are also provided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. Antibodies

The present invention provides agents that specifically bind human VEGFproteins and/or human DLL4 proteins. These agents are referred to hereinas “VEGF/DLL4-binding agents”. The phrase “VEGF/DLL4-binding agent”encompasses agents that bind only VEGF, agents that bind only DLL4, andbispecific agents that bind both VEGF and DLL4. In certain embodiments,in addition to specifically binding VEGF and/or DLL4, theVEGF/DLL4-binding agents further specifically bind at least oneadditional target or antigen. In some embodiments, the VEGF/DLL4-bindingagent is an antibody. In some embodiments, the VEGF/DLL4-binding agentis a polypeptide. In certain embodiments, the VEGF/DLL4-binding agentspecifically binds human VEGF. In certain embodiments, theVEGF/DLL4-binding agent specifically binds human DLL4. In certainembodiments, the VEGF/DLL4-binding agent is a bispecific antibody. Incertain embodiments, the VEGF/DLL4-binding agent is a bispecificantibody that specifically binds human VEGF and human DLL4. Thefull-length amino acid (aa) sequences for human VEGF (VEGF-A) and humanDLL4 are known in the art and are provided herein as SEQ ID NO:27 (VEGF)and SEQ ID NO:23 (DLL4).

In certain embodiments, the VEGF/DLL4-binding agent or antibody bindsVEGF and/or DLL4 with a dissociation constant (K_(D)) of about 1 μM orless, about 100 nM or less, about 40 nM or less, about 20 nM or less,about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. Insome embodiments, a VEGF/DLL4-binding agent or antibody binds VEGFand/or DLL4 with a K_(D) of about 20 nM or less. In some embodiments, aVEGF/DLL4-binding agent or antibody binds VEGF and/or DLL4 with a K_(D)of about 10 nM or less. In some embodiments, a VEGF/DLL4-binding agentor antibody binds VEGF and/or DLL4 with a K_(D) of about 1 nM or less.In some embodiments, a VEGF/DLL4-binding agent or antibody binds VEGFand/or DLL4 with a K_(D) of about 0.1 nM or less. In some embodiments,the VEGF/DLL4-binding agent binds both human VEGF and mouse VEGF with aK_(D) of about 100 nM or less. In some embodiments, theVEGF/DLL4-binding agent binds both human VEGF and mouse VEGF with aK_(D) of about 50 nM or less. In some embodiments, a VEGF/DLL4-bindingagent binds both human DLL4 and mouse DLL4 with a K_(D) of about 100 nMor less. In some embodiments, a VEGF/DLL4-binding agent binds both humanDLL4 and mouse DLL4 with a K_(D) of about 50 nM or less. In someembodiments, the dissociation constant of the binding agent (e.g., anantibody) to VEGF is the dissociation constant determined using a VEGFfusion protein comprising at least a portion of VEGF immobilized on aBiacore chip. In some embodiments, the dissociation constant of thebinding agent (e.g., an antibody) to DLL4 is the dissociation constantdetermined using a DLL4-fusion protein comprising at least a portion ofDLL4 immobilized on a Biacore chip.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a first antigen-binding site that specificallybinds VEGF and a second antigen-binding site that specifically bindsDLL4. In some embodiments, a VEGF/DLL4-binding agent or antibody bindsboth VEGF and DLL4 with a K_(D) of about 100 nM or less. In someembodiments, a VEGF/DLL4-binding agent or antibody binds both VEGF andDLL4 with a K_(D) of about 50 nM or less. In some embodiments, aVEGF/DLL4-binding agent or antibody binds both VEGF and DLL4 with aK_(D) of about 20 nM or less. In some embodiments, a VEGF/DLL4-bindingagent or antibody binds both VEGF and DLL4 with a K_(D) of about 10 nMor less. In some embodiments, a VEGF/DLL4-binding agent or antibodybinds both VEGF and DLL4 with a K_(D) of about 1 nM or less. In someembodiments, the affinity of one of the antigen-binding sites may beweaker than the affinity of the other antigen-binding site. For example,the K_(D) of one antigen binding site may be about 1 nM and the K_(D) ofthe second antigen-binding site may be about 10 nM. In some embodiments,the difference in affinity between the two antigen-binding sites may beabout 2-fold or more, about 3-fold or more, about 5-fold or more, about8-fold or more, about 10-fold or more, about 15-fold or more, about20-fold or more, about 30-fold or more, about 50-fold or more, or about100-fold or more. Modulation of the affinities of the twoantigen-binding sites may affect the biological activity of thebispecific antibody. For example, decreasing the affinity of theantigen-binding site for DLL4 or VEGF, may have a desirable effect, forexample decreased toxicity of the binding agent or increased therapeuticindex.

By way of non-limiting example, the bispecific antibody may comprise (a)a first antigen-binding site that binds human VEGF with a K_(D) betweenabout 0.1 nM and about 1.0 nM, and (b) a second antigen-binding sitethat specifically binds human DLL4 with a K_(D) between about 0.1 nM andabout 20 nM, between about 0.5 nM and about 20 nM, between about 1.0 nMand 10 nM. In certain embodiments, the bispecific antibody comprises twoidentical light chains.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., an antibody)binds VEGF and/or DLL4 with a half maximal effective concentration(EC₅₀) of about 1 μM or less, about 100 nM or less, about 40 nM or less,about 20 nM or less, about 10 nM or less, about 1 nM or less, or about0.1 nM or less. In certain embodiments, a VEGF/DLL4-binding agent (e.g.,an antibody) binds VEGF and/or DLL4 with a half maximal effectiveconcentration (EC₅₀) of about 1 μM or less, about 100 nM or less, about40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM orless, or about 0.1 nM or less.

In certain embodiments, the VEGF/DLL4-binding agent is an antibody. Insome embodiments, the antibody is a recombinant antibody. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a chimeric antibody. In some embodiments, the antibodyis a humanized antibody. In some embodiments, the antibody is a humanantibody. In certain embodiments, the antibody is an IgA, IgD, IgE, IgG,or IgM antibody. In certain embodiments, the antibody is an IgG1antibody. In certain embodiments, the antibody is an IgG2 antibody. Incertain embodiments, the antibody is an antibody fragment comprising anantigen-binding site. In some embodiments, the antibody is a bispecificantibody. In some embodiments, the antibody is monovalent, monospecific,bivalent, or multispecific. In some embodiments, the antibody isconjugated to a cytotoxic moiety. In some embodiments, the antibody isisolated. In some embodiments, the antibody is substantially pure.

The VEGF/DLL4-binding agents (e.g., antibodies) of the present inventioncan be assayed for specific binding by any method known in the art. Theimmunoassays which can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such asBiacore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA,“sandwich” immunoassay, immunoprecipitation assay, precipitationreaction, gel diffusion precipitin reaction, immunodiffusion assay,agglutination assay, complement-fixation assay, immunoradiometric assay,fluorescent immunoassay, homogeneous time-resolved fluorescence assay(HTRF), and protein A immunoassay. Such assays are routine andwell-known in the art (see, e.g., Ausubel et al., Editors, 1994-present,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, N.Y.).

For example, the specific binding of an antibody to human VEGF and/orhuman DLL4 may be determined using ELISA. An ELISA assay comprisespreparing antigen, coating wells of a 96 well microtiter plate withantigen, adding the antibody or other binding agent conjugated to adetectable compound such as an enzymatic substrate (e.g. horseradishperoxidase or alkaline phosphatase) to the well, incubating for a periodof time, and detecting the presence of the binding agent bound to theantigen. In some embodiments, the binding agent or antibody is notconjugated to a detectable compound, but instead a second antibody thatrecognizes the binding agent or antibody (e.g., an anti-Fc antibody) andis conjugated to a detectable compound is added to the well. In someembodiments, instead of coating the well with the antigen, the bindingagent or antibody can be coated to the well and a second antibodyconjugated to a detectable compound can be added following the additionof the antigen to the coated well. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thesignal detected as well as other variations of ELISAs known in the art.

In another example, the specific binding of an antibody to human VEGFand/or human DLL4 may be determined using FACS. A FACS screening assaymay comprise generating a cDNA construct that expresses an antigen as afusion protein, transfecting the construct into cells, expressing theantigen on the surface of the cells, mixing the binding agent orantibody with the transfected cells, and incubating for a period oftime. The cells bound by the binding agent or antibody may be identifiedby using a secondary antibody conjugated to a detectable compound (e.g.,PE-conjugated anti-Fc antibody) and a flow cytometer. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto optimize the signal detected as well as other variations of FACS thatmay enhance screening (e.g., screening for blocking antibodies).

The binding affinity of an antibody or other binding-agent to an antigen(e.g., VEGF or DLL4) and the off-rate of an antibody-antigen interactioncan be determined by competitive binding assays. One example of acompetitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I), or fragment or variantthereof, with the antibody of interest in the presence of increasingamounts of unlabeled antigen followed by the detection of the antibodybound to the labeled antigen. The affinity of the antibody for theantigen and the binding off-rates can be determined from the data byScatchard plot analysis. In some embodiments, Biacore kinetic analysisis used to determine the binding on and off rates of antibodies oragents that bind an antigen (e.g., VEGF or DLL4). Biacore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized antigen (e.g., VEGF or DLL4) on theirsurface.

In certain embodiments, the invention provides a VEGF-binding agent(e.g., an antibody) that specifically binds human VEGF, wherein theVEGF-binding agent (e.g., an antibody) comprises one, two, three, four,five, and/or six of the CDRs of antibody 219R45 (see Table 1). In someembodiments, the VEGF-binding agent comprises one or more of the CDRs of219R45, two or more of the CDRs of 219R45, three or more of the CDRs of219R45, four or more of the CDRs of 219R45, five or more of the CDRs of219R45, or all six of the CDRs of 219R45. In some embodiments, theVEGF-binding agent binds human VEGF and mouse VEGF.

TABLE 1 219R45 HC CDR1 NYWMH (SEQ ID NO: 17) HC CDR2 DINPSNGRTSYKEKFKR(SEQ ID NO: 18) HC CDR3 HYDDKYYPLMDY (SEQ ID NO: 19) LC CDR1RASESVDNYGISFMK (SEQ ID NO: 20) LC CDR2 AASNQGS (SEQ ID NO: 21) LC CDR3QQSKEVPWTFGG (SEQ ID NO: 22)

In certain embodiments, the invention provides a VEGF-binding agent(e.g., an antibody) that specifically binds human VEGF, wherein theVEGF-binding agent comprises a heavy chain CDR1 comprising NYWMH (SEQ IDNO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18),and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19). In someembodiments, the VEGF-binding agent further comprises a light chain CDR1comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprisingAASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG(SEQ ID NO:22). In certain embodiments, the VEGF-binding agentcomprises: (a) a heavy chain CDR1 comprising NYWMH (SEQ ID NO:17), aheavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and aheavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19), and (b) a lightchain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the invention provides a VEGF-binding agent(e.g., an antibody) that specifically binds human VEGF, wherein theVEGF-binding agent comprises: (a) a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (b) a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQID NO:18), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (c) a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ IDNO:19), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ IDNO:20), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (e) a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and (f) a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

In certain embodiments, the invention provides a VEGF-binding agent(e.g., an antibody) that specifically binds VEGF, wherein theVEGF-binding agent comprises a heavy chain variable region having atleast about 80% sequence identity to SEQ ID NO:11, and a light chainvariable region having at least 80% sequence identity to SEQ ID NO:12.In certain embodiments, the VEGF-binding agent comprises a heavy chainvariable region having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:11. In certain embodiments, the VEGF-binding agentcomprises a light chain variable region having at least about 85%, atleast about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:12. In certain embodiments, theVEGF-binding agent comprises a heavy chain variable region having atleast about 95% sequence identity to SEQ ID NO:11, and a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the VEGF-binding agent comprises a heavychain variable region comprising SEQ ID NO:11, and a light chainvariable region comprising SEQ ID NO:12. In certain embodiments, theVEGF-binding agent comprises a heavy chain variable region consistingessentially of SEQ ID NO:11, and a light chain variable regionconsisting essentially of SEQ ID NO:12. In some embodiments, theVEGF-binding agent comprises a heavy chain comprising SEQ ID NO:49, anda light chain comprising SEQ ID NO:8. In some embodiments, theVEGF-binding antibody or other agent comprises a heavy chain comprisingSEQ ID NO:7, and a light chain comprising SEQ ID NO:8.

In some embodiments, the VEGF-binding agent binds VEGF with a K_(D) ofabout 10 nM or less. In some embodiments, the VEGF-binding agent bindsVEGF with a K_(D) of about 1 nM or less. In some embodiments, theVEGF-binding agent binds VEGF with a K_(D) of about 0.1 nM or less. Insome embodiments, the VEGF-binding agent binds VEGF with a K_(D) ofabout 0.01 nM or less. In some embodiments, at least one amino acidresidue in at least one CDR of the VEGF-binding agent is substitutedwith a different amino acid so that the affinity of the VEGF-bindingagent for VEGF is altered. In some embodiments, the affinity of theVEGF-binding agent is increased. In some embodiments, the affinity ofthe VEGF-binding agent is decreased. In some embodiments, theVEGF-binding agent binds human VEGF. In some embodiments, theVEGF-binding agent binds human VEGF and mouse VEGF.

In certain embodiments, the VEGF-binding agent comprises the heavy chainvariable region and light chain variable region of the 219R45 antibody.In certain embodiments, the VEGF-binding agent comprises the heavy chainand light chain of the 219R45 antibody (with or without the leadersequence). In certain embodiments, a VEGF-binding agent is the 219R45antibody.

In certain embodiments, a VEGF-binding agent comprises, consistsessentially of, or consists of, the antibody 219R45.

In certain embodiments, a VEGF-binding agent (e.g., an antibody) bindsthe same epitope, or essentially the same epitope, on VEGF as anantibody of the invention. In another embodiment, a VEGF-binding agentis an antibody that binds an epitope on VEGF that overlaps with theepitope on VEGF bound by an antibody of the invention. In certainembodiments, a VEGF-binding agent (e.g., an antibody) binds the sameepitope, or essentially the same epitope, on VEGF as antibody 219R45. Inanother embodiment, the VEGF-binding agent is an antibody that binds anepitope on VEGF that overlaps with the epitope on VEGF bound by antibody219R45.

In some embodiments, the VEGF-binding agent inhibits binding of VEGF toat least one VEGF receptor. In certain embodiments, the VEGF-bindingagent inhibits binding of human VEGF to VEGFR-1 or VEGFR-2. In someembodiments, the VEGF-binding agent specifically binds VEGF andmodulates angiogenesis. In some embodiments, the VEGF-binding agentspecifically binds VEGF and inhibits angiogenesis. In some embodiments,the VEGF-binding agent specifically binds VEGF and inhibits tumorgrowth.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent (e.g., an antibody) comprises one, two, three, four,five, and/or six of the CDRs of antibody 21R79 (see Table 2). In someembodiments, the DLL4-binding agent comprises one or more of the CDRs of21R79, two or more of the CDRs of 21R79, three or more of the CDRs of21R79, four or more of the CDRs of 21R79, five or more of the CDRs of21R79, or all six of the CDRs of 21R79. In certain embodiments, theinvention provides a DLL4-binding agent (e.g., an antibody) thatspecifically binds human DLL4, wherein the DLL4-binding agent (e.g., anantibody) comprises one, two, three, four, five, and/or six of the CDRsof antibody 21R75 (see Table 2). In some embodiments, the DLL4-bindingagent comprises one or more of the CDRs of 21R75, two or more of theCDRs of 21R75, three or more of the CDRs of 21R75, four or more of theCDRs of 21R75, five or more of the CDRs of 21R75, or all six of the CDRsof 21R75. In certain embodiments, the invention provides a DLL4-bindingagent (e.g., an antibody) that specifically binds human DLL4, whereinthe DLL4-binding agent (e.g., an antibody) comprises one, two, three,four, five, and/or six of the CDRs of antibody 21R83 (see Table 2). Insome embodiments, the DLL4-binding agent comprises one or more of theCDRs of 21R83, two or more of the CDRs of 21R83, three or more of theCDRs of 21R83, four or more of the CDRs of 21R83, five or more of theCDRs of 21R83, or all six of the CDRs of 21R83. In some embodiments, theDLL4-binding agent binds human DLL4 and mouse DLL4.

TABLE 2 21R79 21R75 21R83 HC CDR1 TAYYIH TAYYIH TAYYIH (SEQ ID NO: 13)(SEQ ID NO: 13) (SEQ ID NO: 13) HC CDR2 YIANYNRATNYNQKFKGYIAGYKDATNYNQKFKG YISNYNRATNYNQKFKG (SEQ ID NO: 14) (SEQ ID NO: 59)(SEQ ID NO: 65) HC CDR3 RDYDYDVGMDY RDYDYDVGMDY RDYDYDVGMDY(SEQ ID NO: 16) (SEQ ID NO: 16) (SEQ ID NO: 16) LC CDR1 RASESVDNYGISFMKRASESVDNYGISFMK RASESVDNYGISFMK (SEQ ID NO: 20) (SEQ ID NO: 20)(SEQ ID NO: 20) LC CDR2 AASNQGS AASNQGS AASNQGS (SEQ ID NO: 21)(SEQ ID NO: 21) (SEQ ID NO: 21) LC CDR3 QQSKEVPWTFGG QQSKEVPWTFGGQQSKEVPWTFGG (SEQ ID NO: 22) (SEQ ID NO: 22) (SEQ ID NO: 22)

In certain embodiments, the heavy chain CDR1 of the DLL4-bindingantibody is a minimal HC CDR1 comprising AYYIH (SEQ ID NO:79).

In some embodiments, the binding agent is an antibody that binds humanDLL4 and comprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13)or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprisingYIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁ is serine or alanine,X₂ is serine, asparagine, or glycine, X₃ is asparagine or lysine, and X₄is glysine, arginine, or aspartic acid, and a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:16); and a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ IDNO:14), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). Insome embodiments, the DLL4-binding agent further comprises a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4-bindingagent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ IDNO:13), a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH(SEQ ID NO:13), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (b) a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQID NO:14), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (c) a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ IDNO:16), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ IDNO:20), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (e) a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and (f) a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds DLL4, wherein theDLL4-binding agent comprises a heavy chain variable region having atleast about 80% sequence identity to SEQ ID NO:10, and a light chainvariable region having at least 80% sequence identity to SEQ ID NO:12.In certain embodiments, the DLL4-binding agent comprises a heavy chainvariable region having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:10. In certain embodiments, the DLL4-binding agentcomprises a light chain variable region having at least about 85%, atleast about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region having atleast about 95% sequence identity to SEQ ID NO:10, and a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the DLL4-binding agent comprises a heavychain variable region comprising SEQ ID NO:10, and a light chainvariable region comprising SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region consistingessentially of SEQ ID NO:10, and a light chain variable regionconsisting essentially of SEQ ID NO:12. In some embodiments, theDLL4-binding agent comprises a heavy chain comprising SEQ ID NO:48, anda light chain comprising SEQ ID NO:8. In some embodiments, theDLL4-binding antibody or other agent comprises a heavy chain comprisingSEQ ID NO:6, and a light chain comprising SEQ ID NO:8. In someembodiments, the antibody is a bispecific antibody.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ IDNO:59), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). Insome embodiments, the DLL4-binding agent further comprises a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4-bindingagent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ IDNO:13), a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH(SEQ ID NO:13), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (b) a heavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQID NO:59), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (c) a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ IDNO:16), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ IDNO:20), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (e) a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and (f) a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds DLL4, wherein theDLL4-binding agent comprises a heavy chain variable region having atleast about 80% sequence identity to SEQ ID NO:58, and a light chainvariable region having at least 80% sequence identity to SEQ ID NO:12.In certain embodiments, the DLL4-binding agent comprises a heavy chainvariable region having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:58. In certain embodiments, the DLL4-binding agentcomprises a light chain variable region having at least about 85%, atleast about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region having atleast about 95% sequence identity to SEQ ID NO:58, and a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the DLL4-binding agent comprises a heavychain variable region comprising SEQ ID NO:58, and a light chainvariable region comprising SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region consistingessentially of SEQ ID NO:58, and a light chain variable regionconsisting essentially of SEQ ID NO:12. In some embodiments, theDLL4-binding agent comprises a heavy chain comprising SEQ ID NO:56, anda light chain comprising SEQ ID NO:8.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ IDNO:65), and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). Insome embodiments, the DLL4-binding agent further comprises a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In certain embodiments, the DLL4-bindingagent comprises: (a) a heavy chain CDR1 comprising TAYYIH (SEQ IDNO:13), a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16), and (b) alight chain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a lightchain CDR2 comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3comprising QQSKEVPWTFGG (SEQ ID NO:22).

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds human DLL4, wherein theDLL4-binding agent comprises: (a) a heavy chain CDR1 comprising TAYYIH(SEQ ID NO:13), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (b) a heavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQID NO:65), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (c) a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ IDNO:16), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (d) a light chain CDR1 comprising RASESVDNYGISFMK (SEQ IDNO:20), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (e) a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;and (f) a light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

In certain embodiments, the invention provides a DLL4-binding agent(e.g., an antibody) that specifically binds DLL4, wherein theDLL4-binding agent comprises a heavy chain variable region having atleast about 80% sequence identity to SEQ ID NO:64, and a light chainvariable region having at least 80% sequence identity to SEQ ID NO:12.In certain embodiments, the DLL4-binding agent comprises a heavy chainvariable region having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:64. In certain embodiments, the DLL4-binding agentcomprises a light chain variable region having at least about 85%, atleast about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region having atleast about 95% sequence identity to SEQ ID NO:64, and a light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the DLL4-binding agent comprises a heavychain variable region comprising SEQ ID NO:64, and a light chainvariable region comprising SEQ ID NO:12. In certain embodiments, theDLL4-binding agent comprises a heavy chain variable region consistingessentially of SEQ ID NO:64, and a light chain variable regionconsisting essentially of SEQ ID NO:12. In some embodiments, theDLL4-binding agent comprises a heavy chain comprising SEQ ID NO:62, anda light chain comprising SEQ ID NO:8. In some embodiments, the agent isa bispecific antibody.

In some embodiments, the DLL4-binding agent is an antibody thatcomprises a heavy chain comprising SEQ ID NO:5, and a light chaincomprising SEQ ID NO:8. In some embodiments, the antibody is abispecific antibody.

In some embodiments, the DLL4-binding agent binds DLL4 with a K_(D) of25 nM or less. In some embodiments, the DLL4-binding agent binds DLL4with a K_(D) of 10 nM or less. In some embodiments, the DLL4-bindingagent binds DLL4 with a K_(D) of about 1 nM or less. In someembodiments, the DLL4-binding agent binds DLL4 with a K_(D) of about 0.1nM or less. In some embodiments, the DLL4-binding agent binds DLL4 witha K_(D) of about 0.01 nM or less. In some embodiments, at least oneamino acid residue in at least one CDR of the DLL4-binding agent issubstituted with a different amino acid so that the affinity of theDLL4-binding agent for DLL4 is altered. In some embodiments, theaffinity of the DLL4-binding agent is increased. In some embodiments,the affinity of the DLL4-binding agent is decreased.

In certain embodiments, the DLL4-binding agent comprises the heavy chainvariable region and the light chain variable region of the 21R79antibody. In certain embodiments, the DLL4-binding agent comprises theheavy chain and light chain of the 21R79 antibody (with or without theleader sequence). In certain embodiments, the DLL4-binding agent is the21R79 antibody.

In certain embodiments, a DLL4-binding agent comprises, consistsessentially of, or consists of, the antibody 21R79.

In certain embodiments, the DLL4-binding agent comprises the heavy chainvariable region and the light chain variable region of the 21R75antibody. In certain embodiments, the DLL4-binding agent comprises theheavy chain and light chain of the 21R75 antibody (with or without theleader sequence). In certain embodiments, the DLL4-binding agent is the21R75 antibody.

In certain embodiments, a DLL4-binding agent comprises, consistsessentially of, or consists of, the antibody 21R75.

In certain embodiments, the DLL4-binding agent comprises the heavy chainvariable region and the light chain variable region of the 21R83antibody. In certain embodiments, the DLL4-binding agent comprises theheavy chain and light chain of the 21R83 antibody (with or without theleader sequence). In certain embodiments, the DLL4-binding agent is the21R83 antibody.

In certain embodiments, a DLL4-binding agent comprises, consistsessentially of, or consists of, the antibody 21R83.

In some embodiments, a DLL4-binding agent binds an N-terminal fragmentof human DLL4 (amino acids 1-191 of SEQ ID NO:24). In some embodiments,the DLL4-binding agent binds an epitope comprising amino acids 40-47 ofSEQ ID NO:25. In some embodiments, the DLL4-binding agent binds anepitope comprising amino acids 113-120 of SEQ ID NO:25. In someembodiments, the DLL4-binding agent binds an epitope comprising aminoacids 40-47 of SEQ ID NO:25 and amino acids 113-120 of SEQ ID NO:25.

In certain embodiments, a DLL4-binding agent (e.g., an antibody) bindsthe same epitope, or essentially the same epitope, on DLL4 as anantibody of the invention. In another embodiment, a DLL4-binding agentis an antibody that binds an epitope on DLL4 that overlaps with theepitope on DLL4 bound by an antibody of the invention. In certainembodiments, a DLL4-binding agent (e.g., an antibody) binds the sameepitope, or essentially the same epitope, on DLL4 as antibody 21R79. Inanother embodiment, the DLL4-binding agent is an antibody that binds anepitope on DLL4 that overlaps with the epitope on DLL4 bound by antibody21R79. In certain embodiments, a DLL4-binding agent (e.g., an antibody)binds the same epitope, or essentially the same epitope, on DLL4 asantibody 21R75. In another embodiment, the DLL4-binding agent is anantibody that binds an epitope on DLL4 that overlaps with the epitope onDLL4 bound by antibody 21R75. In certain embodiments, a DLL4-bindingagent (e.g., an antibody) binds the same epitope, or essentially thesame epitope, on DLL4 as antibody 21R83. In another embodiment, theDLL4-binding agent is an antibody that binds an epitope on DLL4 thatoverlaps with the epitope on DLL4 bound by antibody 21R83.

In some embodiments, the DLL4-binding agent inhibits binding of DLL4 toat least one Notch receptor. In certain embodiments, the Notch receptoris Notch1, Notch2, Notch3, or Notch4. In some embodiments, theDLL4-binding agent specifically binds DLL4 and inhibits DLL4 activity.In some embodiments, the DLL4-binding agent specifically binds DLL4 andinhibits Notch signaling. In some embodiments, the DLL4-binding agentspecifically binds DLL4 and modulates angiogenesis. In some embodiments,the DLL4-binding agent specifically binds DLL4 and inhibits tumorgrowth. In some embodiments, the DLL4-binding agent specifically bindsDLL4 and inhibits tumorigenicity. In some embodiments, the DLL4-bindingagent specifically binds DLL4 and reduces the number or frequency ofCSCs in a tumor.

In certain embodiments, the invention provides a VEGF/DLL4-binding agentthat is a bispecific antibody. In some embodiments, the VEGF/DLL4binding agent is a bispecific antibody comprising a firstantigen-binding site that specifically binds human VEGF. In someembodiments, the VEGF/DLL4 binding agent is a bispecific antibodycomprising a first antigen-binding site that specifically binds humanVEGF and a second antigen-binding site that binds a tumor-associatedtarget. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising: a first antigen-binding site that specificallybinds human VEGF, wherein the first antigen-binding site comprises aheavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19). In some embodiments, thebispecific antibody further comprises: a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising: a first antigen-binding site that specificallybinds human VEGF, wherein the first antigen-binding site comprises (a) aheavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19), and (b) a light chain CDR1comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprisingAASNQGS (SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG(SEQ ID NO:22).

In some embodiments, the VEGF/DLL4 binding agent is a bispecificantibody comprising a first heavy chain variable region having at leastabout 80% sequence identity to SEQ ID NO:11. In some embodiments, thebispecific antibody further comprises a light chain variable regionhaving at least 80% sequence identity to SEQ ID NO:12. In certainembodiments, the bispecific VEGF/DLL4-binding agent comprises a firstheavy chain variable region having at least about 85%, at least about90%, at least about 95%, at least about 97%, or at least about 99%sequence identity to SEQ ID NO:11, and a light chain variable regionhaving at least about 85%, at least about 90%, at least about 95%, atleast about 97%, or at least about 99% sequence identity to SEQ IDNO:12.

In certain embodiments, the invention provides a VEGF/DLL4-binding agentthat is a bispecific antibody. In some embodiments, the VEGF/DLL4binding agent is a bispecific antibody comprising a firstantigen-binding site that specifically binds human DLL4. In someembodiments, the VEGF/DLL4 binding agent is a bispecific antibodycomprising a first antigen-binding site that specifically binds humanDLL4 and a second antigen-binding site that binds a tumor-associatedtarget. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising: a first antigen-binding site that specificallybinds human DLL4, wherein the first antigen-binding site comprises aheavy chain CDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ IDNO:79), a heavy chain CDR2 comprising YIX₁X₂YX₃X₄ATNYNQKFKG (SEQ IDNO:80), wherein X₁ is serine or alanine, X₂ is serine, asparagine, orglycine, X₃ is asparagine or lysine, and X₄ is glysine, arginine, oraspartic acid, and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ IDNO:16); and a light chain CDR1 comprising RASESVDNYGISFMK (SEQ IDNO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21), and alight chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22). In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibodycomprising: a first antigen-binding site that specifically binds humanDLL4, wherein the first antigen-binding site comprises a heavy chainCDR1 comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIANYNRATNYNQKFKG (SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15),YIAGYKDATNYNQKFKG (SEQ ID NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In someembodiments, the bispecific antibody comprises a first antigen-bindingsite comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), aheavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14), and aheavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In someembodiments, the bispecific antibody comprises a first antigen-bindingsite comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), aheavy chain CDR2 comprising YISSYNGATNYNQKFKG (SEQ ID NO:15), and aheavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In someembodiments, the bispecific antibody comprises a first antigen-bindingsite comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), aheavy chain CDR2 comprising YIAGYKDATNYNQKFKG (SEQ ID NO:59), and aheavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In someembodiments, the bispecific antibody comprises a first antigen-bindingsite comprising a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), aheavy chain CDR2 comprising YISNYNRATNYNQKFKG (SEQ ID NO:65), and aheavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16). In someembodiments, the bispecific antibody further comprises: a light chainCDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody comprising: a first antigen-binding sitethat specifically binds human DLL4, wherein the first antigen-bindingsite comprises (a) a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13),a heavy chain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14),YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), orYIANYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16), and (b) a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).

In some embodiments, the VEGF/DLL4 binding agent is a bispecificantibody comprising a first heavy chain variable region having at leastabout 80% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58,or SEQ ID NO:64. In some embodiments, the bispecific antibody furthercomprises a light chain variable region having at least 80% sequenceidentity to SEQ ID NO:12. In certain embodiments, the bispecificVEGF/DLL4-binding agent comprises a first heavy chain variable regionhaving at least about 85%, at least about 90%, at least about 95%, atleast about 97%, or at least about 99% sequence identity to SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64; and/or a light chainvariable region having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NO:12.

In certain embodiments, the invention provides a VEGF/DLL4-binding agent(e.g., a bispecific antibody) that specifically binds human VEGF andhuman DLL4. In some embodiments, the bispecific antibody comprises: a) afirst antigen-binding site that specifically binds human VEGF, and b) asecond antigen-binding site that specifically binds human DLL4, whereinthe first antigen-binding site comprises a heavy chain CDR1 comprisingNYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR(SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ IDNO:19); wherein the second antigen-binding site comprises a heavy chainCDR1 comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavychain CDR2 comprising YIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁is serine or alanine, X₂ is serine, asparagine, or glycine, X₃ isasparagine or lysine, and X₄ is glysine, arginine, or aspartic acid, anda heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and a lightchain CDR1 comprising RASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2comprising AASNQGS (SEQ ID NO:21), and a light chain CDR3 comprisingQQSKEVPWTFGG (SEQ ID NO:22). In some embodiments, a bispecific antibodycomprises a first antigen-binding site that specifically binds humanVEGF, and a second antigen-binding site that specifically binds humanDLL4, wherein the first antigen-binding site comprises a heavy chainCDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprisingDINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 comprisingHYDDKYYPLMDY (SEQ ID NO:19), and the second antigen-binding sitecomprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavychain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14),YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), orYISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).

In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF, and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R79.

In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF, and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYISSYNGATNYNQKFKG (SEQ ID NO:15), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody is 219R45-MB-21M18.

In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF, and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site which comprises a heavy chain CDR1 comprisingNYWMH (SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR(SEQ ID NO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ IDNO:19), and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R75.

In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF, and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody is 219R45-MB-21R83.

In some embodiments, the VEGF/DLL4 binding agent (e.g., a bispecificantibody) comprises a first heavy chain variable region having at leastabout 80% sequence identity to SEQ ID NO:11, a second heavy chainvariable region having at least about 80% sequence identity to SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64, and a first and asecond light chain variable region having at least 80% sequence identityto SEQ ID NO:12. In certain embodiments, the bispecificVEGF/DLL4-binding agent comprises a first heavy chain variable regionhaving at least about 85%, at least about 90%, at least about 95%, atleast about 97%, or at least about 99% sequence identity to SEQ IDNO:11; a second heavy chain variable region having at least about 85%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% sequence identity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58,or SEQ ID NO:64; and a first and a second light chain variable regionhaving at least about 85%, at least about 90%, at least about 95%, atleast about 97%, or at least about 99% sequence identity to SEQ IDNO:12. In certain embodiments, the bispecific VEGF/DLL4-binding agentcomprises a first heavy chain variable region having at least about 95%sequence identity to SEQ ID NO:11, a second heavy chain variable regionhaving at least about 95% sequence identity to SEQ ID NO:9, and a firstand a second light chain variable region having at least about 95%sequence identity to SEQ ID NO:12. In certain embodiments, thebispecific VEGF/DLL4-binding agent comprises a first heavy chainvariable region having at least about 95% sequence identity to SEQ IDNO:11, a second heavy chain variable region having at least about 95%sequence identity to SEQ ID NO:10, and a first and a second light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the bispecific VEGF/DLL4-binding agentcomprises a first heavy chain variable region having at least about 95%sequence identity to SEQ ID NO:11, a second heavy chain variable regionhaving at least about 95% sequence identity to SEQ ID NO:58, and a firstand a second light chain variable region having at least about 95%sequence identity to SEQ ID NO:12. In certain embodiments, thebispecific VEGF/DLL4-binding agent comprises a first heavy chainvariable region having at least about 95% sequence identity to SEQ IDNO:11, a second heavy chain variable region having at least about 95%sequence identity to SEQ ID NO:64, and a first and a second light chainvariable region having at least about 95% sequence identity to SEQ IDNO:12. In certain embodiments, the bispecific VEGF/DLL4-binding agentcomprises a first heavy chain variable region comprising SEQ ID NO:11, asecond heavy chain variable region comprising SEQ ID NO:9, and a firstand a second light chain variable region comprising SEQ ID NO:12. Incertain embodiments, the bispecific VEGF/DLL4-binding agent comprises afirst heavy chain variable region comprising SEQ ID NO:11, a secondheavy chain variable region comprising SEQ ID NO:10, and a first and asecond light chain variable region comprising SEQ ID NO:12. In certainembodiments, the bispecific VEGF/DLL4-binding agent comprises a firstheavy chain variable region comprising SEQ ID NO:11, a second heavychain variable region comprising SEQ ID NO:58, and a first and a secondlight chain variable region comprising SEQ ID NO:12. In certainembodiments, the bispecific VEGF/DLL4-binding agent comprises a firstheavy chain variable region comprising SEQ ID NO:11, a second heavychain variable region comprising SEQ ID NO:64, and a first and a secondlight chain variable region comprising SEQ ID NO:12. In certainembodiments, the bispecific VEGF/DLL4-binding agent comprises a firstheavy chain variable region consisting essentially of SEQ ID NO:11, asecond heavy chain variable region consisting essentially of SEQ IDNO:9, and a first and a second light chain variable region consistingessentially of SEQ ID NO:12. In certain embodiments, the bispecificVEGF/DLL4-binding agent comprises a first heavy chain variable regionconsisting essentially of SEQ ID NO:11, a second heavy chain variableregion consisting essentially of SEQ ID NO:10, and a first and a secondlight chain variable region consisting essentially of SEQ ID NO:12. Incertain embodiments, the bispecific VEGF/DLL4-binding agent comprises afirst heavy chain variable region consisting essentially of SEQ IDNO:11, a second heavy chain variable region consisting essentially ofSEQ ID NO:58, and a first and a second light chain variable regionconsisting essentially of SEQ ID NO:12. In certain embodiments, thebispecific VEGF/DLL4-binding agent comprises a first heavy chainvariable region consisting essentially of SEQ ID NO:11, a second heavychain variable region consisting essentially of SEQ ID NO:64, and afirst and a second light chain variable region consisting essentially ofSEQ ID NO:12.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a heavy chain variable region from theanti-VEGF antibody 219R45. In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody which comprises a heavy chain variableregion from the anti-DLL4 antibody 21M18. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody which comprises a heavychain variable region from the anti-DLL4 antibody 21R79. In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibody whichcomprises a heavy chain variable region from the anti-DLL4 antibody21R75. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a heavy chain variable region from theanti-DLL4 antibody 21R83. In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody which comprises a heavy chain variableregion from the anti-VEGF antibody 219R45, a heavy chain variable regionfrom the anti-DLL4 antibody 21R79 and two identical light chain variableregions. In some embodiments, the VEGF/DLL4-binding agent is abispecific antibody which comprises a heavy chain variable region fromthe anti-VEGF antibody 219R45, a heavy chain variable region from theanti-DLL4 antibody 21M18 and two identical light chain variable regions.In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a heavy chain variable region from theanti-VEGF antibody 219R45, a heavy chain variable region from theanti-DLL4 antibody 21R75 and two identical light chain variable regions.In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a heavy chain variable region from theanti-VEGF antibody 219R45, a heavy chain variable region from theanti-DLL4 antibody 21R83 and two identical light chain variable regions.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a first CH3 domain and a second CH3 domain,each of which is modified to promote formation of heteromultimers. Insome embodiments, the first and second CH3 domains are modified using aknobs-into-holes technique. In some embodiments, the first and secondCH3 domains comprise changes in amino acids that result in alteredelectrostatic interactions. In some embodiments, the first and secondCH3 domains comprise changes in amino acids that result in alteredhydrophobic/hydrophilic interactions.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises heavy chain constant regions selected from thegroup consisting of: (a) a first human IgG1 constant region, wherein theamino acids at positions 253 and 292 are substituted with glutamate oraspartate, and a second human IgG1 constant region, wherein the aminoacids at positions 240 and 282 are substituted with lysine; (b) a firsthuman IgG2 constant region, wherein the amino acids at positions 249 and288 are substituted with glutamate or aspartate, and a second human IgG2constant region wherein the amino acids at positions 236 and 278 aresubstituted with lysine; (c) a first human IgG3 constant region, whereinthe amino acids at positions 300 and 339 are substituted with glutamateor aspartate, and a second human IgG3 constant region wherein the aminoacids at positions 287 and 329 are substituted with lysine; and (d) afirst human IgG4 constant region, wherein the amino acids at positions250 and 289 are substituted with glutamate or aspartate, and a secondIgG4 constant region wherein the amino acids at positions 237 and 279are substituted with lysine.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a first human IgG1 constant region with aminoacid substitutions at positions 253 and 292, wherein the amino acids areglutamate or aspartate, and a second human IgG1 constant region withamino acid substitutions at positions 240 and 282, wherein the aminoacids are lysine. In some embodiments, the VEGF/DLL4-binding agent is abispecific antibody which comprises a first human IgG2 constant regionwith amino acid substitutions at positions 249 and 288, wherein theamino acids are glutamate or aspartate, and a second human IgG2 constantregion with amino acid substitutions at positions 236 and 278, whereinthe amino acids are lysine. In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody which comprises a first human IgG3constant region with amino acid substitutions at positions 300 and 339,wherein the amino acids are glutamate or aspartate, and a second humanIgG2 constant region with amino acid substitutions at positions 287 and329, wherein the amino acids are lysine. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody which comprises a firsthuman IgG4 constant region with amino acid substitutions at positions250 and 289, wherein the amino acids are glutamate or aspartate, and asecond human IgG4 constant region with amino acid substitutions atpositions 237 and 279, wherein the amino acids are lysine.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a first human IgG2 constant region with aminoacid substitutions at positions 249 and 288, wherein the amino acids areglutamate, and a second human IgG2 constant region with amino acidsubstitutions at positions 236 and 278, wherein the amino acids arelysine. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a first human IgG2 constant region with aminoacid substitutions at positions 249 and 288, wherein the amino acids areasparate, and a second human IgG2 constant region with amino acidsubstitutions at positions 236 and 278, wherein the amino acids arelysine.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises a heavy chain of SEQ ID NO:7. In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibody whichcomprises a heavy chain of SEQ ID NO:5. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody which comprises a heavychain of SEQ ID NO:56. In some embodiments, the VEGF/DLL4-binding agentis a bispecific antibody which comprises a heavy chain of SEQ ID NO:62.In some embodiments, the bispecific antibody further comprises a lightchain of SEQ ID NO:12. In some embodiments, the VEGF/DLL4-binding agentis a bispecific antibody which comprises a heavy chain of SEQ ID NO:7, aheavy chain of SEQ ID NO:5, and two light chains of SEQ ID NO:8. In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibody whichcomprises a heavy chain of SEQ ID NO:7, a heavy chain of SEQ ID NO:6,and two light chains of SEQ ID NO: 8. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody which comprises a heavychain of SEQ ID NO:7, a heavy chain of SEQ ID NO:56, and two lightchains of SEQ ID NO:8. In some embodiments, the VEGF/DLL4-binding agentis a bispecific antibody which comprises a heavy chain of SEQ ID NO:7, aheavy chain of SEQ ID NO:62, and two light chains of SEQ ID NO:8.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which binds VEGF with a K_(D) of about 50 nM or less, about 25nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM orless. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which binds DLL4 with a K_(D) of about 50 nM or less, about 25nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM orless. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which binds VEGF with a K_(D) of about 50 nM or less and bindsDLL4 with a K_(D) of about 50 nM or less. In some embodiments, thebispecific antibody binds VEGF with a K_(D) of about 25 nM or less andbinds DLL4 with a K_(D) of about 25 nM or less. In some embodiments, thebispecific antibody binds VEGF with a K_(D) of about 10 nM or less andbinds DLL4 with a K_(D) of about 10 nM or less. In some embodiments, thebispecific antibody binds VEGF with a K_(D) of about 1 nM or less andbinds DLL4 with a K_(D) of about 1 nM or less.

In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody which comprises one antigen-binding site with a bindingaffinity that is weaker than the binding affinity of the secondantigen-binding site. For example, in some embodiments, the bispecificantibody may bind VEGF with a K_(D) ranging from about 0.1 nM to 1 nMand may bind DLL4 with a K_(D) ranging from about 1 nM to 10 nM. Or thebispecific antibody may bind VEGF with a K_(D) ranging from about 1 nMto 10 nM and may bind DLL4 with a K_(D) ranging from about 0.1 nM to 1nM. In some embodiments, the bispecific antibody may bind DLL4 with aK_(D) ranging from about 0.1 nM to 1 nM and may bind VEGF with a K_(D)ranging from about 1 nM to 10 nM. Or the bispecific antibody may bindDLL4 with a K_(D) ranging from about 1 nM to 10 nM and may bind VEGFwith a K_(D) ranging from about 0.1 nM to 1 nM In some embodiments, thedifference in affinity between the two antigen-binding sites may beabout 2-fold or more, about 3-fold or more, about 5-fold or more, about8-fold or more, about 10-fold or more, about 15-fold or more, about30-fold or more, about 50-fold or more, or about 100-fold or more. Insome embodiments, at least one amino acid residue in at least one CDR ofthe antigen-binding site for VEGF is substituted with a different aminoacid so that the affinity of the VEGF-binding site is altered. In someembodiments, the affinity of the VEGF-binding site is increased. In someembodiments, the affinity of the VEGF-binding site is decreased. In someembodiments, at least one amino acid residue in at least one CDR of theantigen-binding site for DLL4 is substituted with a different amino acidso that the affinity of the DLL4-binding site is altered. In someembodiments, the affinity of the DLL4-binding site is increased. In someembodiments, the affinity of the DLL4-binding site is decreased. In someembodiments, the affinities of both the VEGF and DLL4 antigen-bindingsites are altered.

The invention provides polypeptides, including but not limited toantibodies, that specifically bind VEGF and/or DLL4. In someembodiments, a polypeptide binds human VEGF. In some embodiments, apolypeptide binds human DLL4. In some embodiments, a polypeptide bindshuman VEGF and mouse VEGF. In some embodiments, a polypeptide bindshuman DLL4 and mouse DLL4.

In some embodiments, a VEGF-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:47, and SEQ ID NO:49.

In some embodiments, a DLL4-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ NO ID:6, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:46, SEQ ID NO:48, SEQID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:63, andSEQ ID NO:64.

In some embodiments, a VEGF/DLL4-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:63, and SEQID NO:64.

In some embodiments, a VEGF/DLL4-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some embodiments, theVEGF/DLL4 binding agent further comprises a polypeptide comprising asequence selected from the group consisting of: SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:11, SEQ ID NO:47, and SEQ ID NO:49. In some embodiments,the VEGF/DLL4 binding agent further comprises a polypeptide comprising asequence selected from the group consisting of: SEQ ID NO:4, SEQ IDNO:8, and SEQ ID NO:12.

In some embodiments, a VEGF/DLL4-binding agent comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:47, and SEQ ID NO:49. In someembodiments, the VEGF/DLL4 binding agent further comprises a polypeptidecomprising a sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some embodiments, theVEGF/DLL4 binding agent further comprises a polypeptide comprising asequence selected from the group consisting of: SEQ ID NO:4, SEQ IDNO:8, and SEQ ID NO:12.

In certain embodiments, a VEGF/DLL4-binding agent (e.g., antibody)competes for specific binding to VEGF with an antibody that comprises aheavy chain variable region comprising SEQ ID NO:11 and a light chainvariable region comprising SEQ ID NO:12. In certain embodiments, aVEGF/DLL4-binding agent competes with antibody 219R45 for specificbinding to human VEGF. In some embodiments, a VEGF/DLL4-binding agent orantibody competes for specific binding to VEGF in an in vitrocompetitive binding assay. In some embodiments, the VEGF is human VEGF.In some embodiments, the VEGF is mouse VEGF.

In certain embodiments, a VEGF-DLL4-binding agent (e.g., an antibody)binds the same epitope, or essentially the same epitope, on VEGF as anantibody of the invention. In another embodiment, a VEGF/DLL4-bindingagent is an antibody that binds an epitope on VEGF that overlaps withthe epitope on VEGF bound by an antibody of the invention. In certainembodiments, a VEGF/DLL4-binding agent (e.g., an antibody) binds thesame epitope, or essentially the same epitope, on VEGF as antibody219R45. In another embodiment, the VEGF/DLL4-binding agent is anantibody that binds an epitope on VEGF that overlaps with the epitope onVEGF bound by antibody 219R45.

In certain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to VEGF with the antibody 219R45 (e.g., ina competitive binding assay).

In certain embodiments, a VEGF/DLL4-binding agent (e.g., antibody)competes for specific binding to DLL4 with an antibody that comprises aheavy chain variable region comprising SEQ ID NO:9 SEQ ID NO:10, SEQ IDNO:58, or SEQ ID NO:64 and a light chain variable region comprising SEQID NO:12. In certain embodiments, a VEGF/DLL4-binding agent competeswith antibody 21R79 for specific binding to human DLL4. In certainembodiments, a VEGF/DLL4-binding agent competes with antibody 21R75 forspecific binding to human DLL4. In certain embodiments, aVEGF/DLL4-binding agent competes with antibody 21R83 for specificbinding to human DLL4. In some embodiments, a VEGF/DLL4-binding agent orantibody competes for specific binding to DLL4 in an in vitrocompetitive binding assay. In some embodiments, the DLL4 is human DLL4.In some embodiments, the DLL4 is mouse DLL4.

In certain embodiments, a VEGF/DLL4-binding agent (e.g., an antibody)binds the same epitope, or essentially the same epitope, on DLL4 as anantibody of the invention. In another embodiment, a VEGF/DLL4-bindingagent is an antibody that binds an epitope on DLL4 that overlaps withthe epitope on DLL4 bound by an antibody of the invention. In certainembodiments, a VEGF/DLL4-binding agent binds the same epitope, oressentially the same epitope, on DLL4 as antibody 21R79. In certainembodiments, a VEGF/DLL4-binding agent binds the same epitope, oressentially the same epitope, on DLL4 as antibody 21R75. In certainembodiments, a VEGF/DLL4-binding agent binds the same epitope, oressentially the same epitope, on DLL4 as antibody 21R83. In anotherembodiment, the VEGF/DLL4-binding agent is an antibody that binds anepitope on DLL4 that overlaps with the epitope on DLL4 bound by antibody21R79. In another embodiment, the VEGF/DLL4-binding agent is an antibodythat binds an epitope on DLL4 that overlaps with the epitope on DLL4bound by antibody 21R75. In another embodiment, the VEGF/DLL4-bindingagent is an antibody that binds an epitope on DLL4 that overlaps withthe epitope on DLL4 bound by antibody 21R83.

In certain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to DLL4 with the antibody 21R79 (e.g., ina competitive binding assay). In certain embodiments, theVEGF/DLL4-binding agent is an agent that competes for specific bindingto DLL4 with the antibody 21R75 (e.g., in a competitive binding assay).In certain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to DLL4 with the antibody 21R83 (e.g., ina competitive binding assay). In certain embodiments, theVEGF/DLL4-binding agent is an agent that competes for specific bindingto DLL4 with the antibody 21M18 (e.g., in a competitive binding assay).

In certain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to VEGF and/or DLL4 with the bispecificantibody 219R45-MB-21M18 (e.g., in a competitive binding assay). Incertain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to VEGF and/or DLL4 with the bispecificantibody 219R45-MB-21M79 (e.g., in a competitive binding assay). Incertain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to VEGF and/or DLL4 with the bispecificantibody 219R45-MB-21M75 (e.g., in a competitive binding assay). Incertain embodiments, the VEGF/DLL4-binding agent is an agent thatcompetes for specific binding to VEGF and/or DLL4 with the bispecificantibody 219R45-MB-21M83 (e.g., in a competitive binding assay).

In certain embodiments, the VEGF/DLL4-binding agent (e.g., an antibody)described herein binds VEGF and modulates VEGF activity. In someembodiments, the VEGF/DLL4-binding agent is a VEGF antagonist andinhibits VEGF activity. In some embodiments, the VEGF/DLL4-binding agentis a VEGF antagonist and modulates angiogenesis. In some embodiments,the VEGF/DLL4-binding agent is a VEGF antagonist and inhibitsangiogenesis. In some embodiments, the VEGF/DLL4-binding agent is a VEGFantagonist and inhibits tumor growth.

In certain embodiments, a VEGF/DLL4-binding agent (e.g., an antibody)described herein binds human DLL4 and modulates DLL4 activity. In someembodiments, a VEGF/DLL4-binding agent is a DLL4 antagonist and inhibitsDLL4 activity. In some embodiments, a VEGF/DLL4-binding agent is a DLL4antagonist and inhibits Notch activity. In some embodiments, aVEGF/DLL4-binding agent is a DLL4 antagonist and inhibits Notchsignaling. In some embodiments, a VEGF/DLL4-binding agent is a DLL4antagonist and modulates angiogenesis. In some embodiments, aVEGF/DLL4-binding agent is a DLL4 antagonist and promotes aberrantangiogenesis. In some embodiments, a VEGF/DLL4-binding agent is a DLL4antagonist and inhibits tumor growth.

In certain embodiments, a VEGF/DLL4-binding agent (e.g., an antibody)described herein is a bispecific antibody that binds human VEGF andmodulates VEGF activity. In certain embodiments, a VEGF/DLL4-bindingagent (e.g., an antibody) described herein is a bispecific antibody thatbinds human DLL4 and modulates DLL4 activity. In certain embodiments, aVEGF/DLL4-binding agent (e.g., an antibody) described herein is abispecific antibody that binds human VEGF and human DLL4 and modulatesboth VEGF and DLL4 activity. In some embodiments, the bispecificantibody is a VEGF antagonist and a DLL4 antagonist and inhibits bothVEGF activity and DLL4 activity. In some embodiments, the bispecificantibody is a VEGF antagonist and a DLL4 antagonist and inhibits VEGFactivity and Notch activity. In some embodiments, the bispecificantibody is a VEGF antagonist and a DLL4 antagonist and inhibits VEGFactivity and Notch signaling. In some embodiments, the bispecificantibody is a VEGF antagonist and a DLL4 antagonist and modulatesangiogenesis. In some embodiments, the bispecific antibody is a VEGFantagonist and a DLL4 antagonist and promotes aberrant angiogenesis. Insome embodiments, the bispecific antibody is a VEGF antagonist and aDLL4 antagonist and inhibits angiogenesis. In some embodiments, thebispecific antibody is a VEGF antagonist and a DLL4 antagonist andinhibits tumor growth.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., an antibodyor a bispecific antibody) is an antagonist of VEGF. In some embodiments,the VEGF/DLL4-binding agent is an antagonist of VEGF and inhibits VEGFactivity. In certain embodiments, the VEGF/DLL4-binding agent inhibitsVEGF activity by at least about 10%, at least about 20%, at least about30%, at least about 50%, at least about 75%, at least about 90%, orabout 100%. In certain embodiments, a VEGF/DLL4-binding agent thatinhibits human VEGF activity is antibody 219R45. In certain embodiments,a VEGF/DLL4-binding agent that inhibits human VEGF activity is abispecific antibody comprising the antigen-binding site of 219R45. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits human VEGFactivity is the bispecific antibody 219R45-MB-21M18. In certainembodiments, a VEGF/DLL4-binding agent that inhibits human VEGF activityis the bispecific antibody 219R45-MB-21R79. In certain embodiments, aVEGF/DLL4-binding agent that inhibits human VEGF activity is thebispecific antibody 219R45-MB-21R75. In certain embodiments, aVEGF/DLL4-binding agent that inhibits human VEGF activity is thebispecific antibody 219R45-MB-21R83.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., an antibody)is an antagonist of DLL4. In some embodiments, the VEGF/DLL4-bindingagent is an antagonist of DLL4 and inhibits DLL4 activity. In certainembodiments, the VEGF/DLL4-binding agent inhibits DLL4 activity by atleast about 10%, at least about 20%, at least about 30%, at least about50%, at least about 75%, at least about 90%, or about 100%. In certainembodiments, a VEGF/DLL4-binding agent that inhibits human DLL4 activityis antibody 21R79. In certain embodiments, a VEGF/DLL4-binding agentthat inhibits human DLL4 activity is antibody 21R75. In certainembodiments, a VEGF/DLL4-binding agent that inhibits human DLL4 activityis antibody 21R83. In certain embodiments, a VEGF/DLL4-binding agentthat inhibits human DLL4 activity is a bispecific antibody comprisingthe antigen-binding site of 21R79. In certain embodiments, aVEGF/DLL4-binding agent that inhibits human DLL4 activity is abispecific antibody comprising the antigen-binding site of 21R75. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits human DLL4activity is a bispecific antibody comprising the antigen-binding site of21R83. In certain embodiments, a VEGF/DLL4-binding agent that inhibitshuman DLL4 activity is the bispecific antibody 219R45-MB-21M18. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits human DLL4activity is the bispecific antibody 219R45-MB-21R79. In certainembodiments, a VEGF/DLL4-binding agent that inhibits human DLL4 activityis the bispecific antibody 219R45-MB-21R75. In certain embodiments, aVEGF/DLL4-binding agent that inhibits human DLL4 activity is thebispecific antibody 219R45-MB-21R83.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., antibody) isan antagonist of Notch signaling. In certain embodiments, theVEGF/DLL4-binding agent inhibits Notch signaling by at least about 10%,at least about 20%, at least about 30%, at least about 50%, at leastabout 75%, at least about 90%, or about 100%. In certain embodiments, aVEGF/DLL4-binding agent that inhibits Notch signaling is antibody 21R79.In certain embodiments, a VEGF/DLL4-binding agent that inhibits Notchsignaling is antibody 21R75. In certain embodiments, a VEGF/DLL4-bindingagent that inhibits Notch signaling is antibody 21R83. In certainembodiments, a VEGF/DLL4-binding agent that inhibits Notch signaling isa bispecific antibody comprising the antigen-binding site of 21R79. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits Notchsignaling is a bispecific antibody comprising the antigen-binding siteof 21R75. In certain embodiments, a VEGF/DLL4-binding agent thatinhibits Notch signaling is a bispecific antibody comprising theantigen-binding site of 21R83. In certain embodiments, aVEGF/DLL4-binding agent that inhibits Notch signaling is the bispecificantibody 219R45-MB-21M18. In certain embodiments, a VEGF/DLL4-bindingagent that inhibits Notch signaling is the bispecific antibody219R45-MB-21R79. In certain embodiments, a VEGF/DLL4-binding agent thatinhibits Notch signaling is the bispecific antibody 219R45-MB-21R75. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits Notchsignaling is the bispecific antibody 219R45-MB-21R83.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., antibody)inhibits binding of VEGF to at least one receptor. In some embodiments,the VEGF/DLL4-binding agent inhibits binding of VEGF to VEGFR-1 orVEGFR-2. In certain embodiments, the VEGF/DLL4-binding agent inhibitsbinding of VEGF to at least one VEGF receptor by at least about 10%, atleast about 25%, at least about 50%, at least about 75%, at least about90%, or at least about 95%. In certain embodiments, a VEGF/DLL4-bindingagent that inhibits binding of human VEGF to at least one VEGF receptoris antibody 219R45. In certain embodiments, a VEGF/DLL4-binding agentthat inhibits binding of human VEGF to at least one VEGF receptor is abispecific antibody comprising the antigen-binding site of 219R45. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits binding ofhuman VEGF to at least one VEGF receptor is the bispecific antibody219R45-MB-21M18. In certain embodiments, a VEGF/DLL4-binding agent thatinhibits binding of human VEGF to at least one VEGF receptor is thebispecific antibody 219R45-MB-21R79. In certain embodiments, aVEGF/DLL4-binding agent that inhibits binding of human VEGF to at leastone VEGF receptor is the bispecific antibody 219R45-MB-21R75. In certainembodiments, a VEGF/DLL4-binding agent that inhibits binding of humanVEGF to at least one VEGF receptor is the bispecific antibody219R45-MB-21R83.

In certain embodiments, the VEGF/DLL4-binding agent (e.g., antibody)inhibits binding of DLL4 protein to at least one Notch receptor. In someembodiments, the VEGF/DLL4-binding agent inhibits binding of DLL4 toNotch1, Notch2, Notch3, and/or Notch4. In certain embodiments, theVEGF/DLL4-binding agent inhibits binding of DLL4 to at least one Notchreceptor by at least about 10%, at least about 25%, at least about 50%,at least about 75%, at least about 90%, or at least about 95%. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits binding ofhuman DLL4 to at least one Notch receptor is antibody 21R79. In certainembodiments, a VEGF/DLL4-binding agent that inhibits binding of humanDLL4 to at least one Notch receptor is antibody 21R75. In certainembodiments, a VEGF/DLL4-binding agent that inhibits binding of humanDLL4 to at least one Notch receptor is antibody 21R83. In certainembodiments, a VEGF/DLL4-binding agent that inhibits binding of humanDLL4 to at least one Notch receptor is a bispecific antibody comprisingthe antigen-binding site of 21R79. In certain embodiments, aVEGF/DLL4-binding agent that inhibits binding of human DLL4 to at leastone Notch receptor is a bispecific antibody comprising theantigen-binding site of 21R75. In certain embodiments, aVEGF/DLL4-binding agent that inhibits binding of human DLL4 to at leastone Notch receptor is a bispecific antibody comprising theantigen-binding site of 21R83. In certain embodiments, aVEGF/DLL4-binding agent that inhibits binding of human DLL4 to at leastone Notch receptor is the bispecific antibody 219R45-MB-21M18. Incertain embodiments, a VEGF/DLL4-binding agent that inhibits binding ofhuman DLL4 to at least one Notch receptor is the bispecific antibody219R45-MB-21R79. In certain embodiments, a VEGF/DLL4-binding agent thatinhibits binding of human DLL4 to at least one Notch receptor is thebispecific antibody 219R45-MB-21R75. In certain embodiments, aVEGF/DLL4-binding agent that inhibits binding of human DLL4 to at leastone Notch receptor is the bispecific antibody 219R45-MB-21R83.

In vivo and in vitro assays for determining whether a VEGF/DLL4-bindingagent (or candidate VEGF/DLL4-binding agent) inhibits VEGF or affectsangiogenesis are known in the art. In vitro assays of angiogenesisinclude but are not limited to, HUVEC proliferation assays, endothelialcell tube formation assays, sprouting (or sprout formation) assays,HUVEC cell migration assays, and invasion assays. In some embodiments,cells in the presence of VEGF and the presence of a VEGF/DLL4-bindingagent are compared to cells in the presence of VEGF without theVEGF/DLL4-binding agent present, and evaluated for effects onangiogenesis (or biological effects associated with angiogenesis). Invivo assays of angiogenesis include, but are not limited to, matrigelplug assays, corneal micropocket assays, and chicken chorioallantoicmembrane (CAM) assays.

In vivo and in vitro assays for determining whether a VEGF/DLL4-bindingagent (or candidate VEGF/DLL4-binding agent) inhibits Notch activationor signaling are known in the art. For example, cell-based, luciferasereporter assays utilizing a TCF/Luc reporter vector containing multiplecopies of the TCF-binding domain upstream of a firefly luciferasereporter gene may be used to measure Notch signaling levels in vitro(Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore,Billerica Mass.). In some embodiments, a cell-based, luciferase reporterassay utilizing a CBF/Luc reporter vector containing multiple copies ofthe CBF-binding domain upstream of a firefly luciferase report genes maybe used. The level of Notch signaling in the presence of one or moreNotch ligands (e.g., DLL4 expressed on the surface of transfected cellsor soluble DLL4-Fc fusion protein) and in the presence of aVEGF/DLL4-binding agent is compared to the level of Notch signalingwithout the VEGF/DLL4-binding agent present.

In certain embodiments, the VEGF/DLL4-binding agents have one or more ofthe following effects: inhibit proliferation of tumor cells, inhibittumor growth, reduce the tumorigenicity of a tumor, reduce the frequencyof cancer stem cells in a tumor, trigger cell death of tumor cells,prevent metastasis of tumor cells, decrease survival of tumor cells,modulate angiogenesis, inhibit angiogenesis, inhibit productiveangiogenesis, or promote aberrant angiogenesis.

In certain embodiments, the VEGF/DLL4-binding agents are capable ofinhibiting tumor growth. In certain embodiments, the VEGF/DLL4-bindingagents are capable of inhibiting tumor growth in vivo (e.g., in axenograft mouse model, and/or in a human having cancer). In certainembodiments, tumor growth is inhibited at least about two-fold, aboutthree-fold, about five-fold, about ten-fold, about 50-fold, about100-fold, or about 1000-fold as compared to an untreated tumor.

In certain embodiments, the VEGF/DLL4-binding agents are capable ofreducing the tumorigenicity of a tumor. In certain embodiments, theVEGF/DLL4-binding agent or antibody is capable of reducing thetumorigenicity of a tumor comprising cancer stem cells in an animalmodel, such as a mouse xenograft model. In certain embodiments, theVEGF/DLL4-binding agent or antibody is capable of reducing thetumorigenicity of a tumor by decreasing the number or frequency ofcancer stem cells in the tumor. In certain embodiments, the number orfrequency of cancer stem cells in a tumor is reduced by at least abouttwo-fold, about three-fold, about five-fold, about ten-fold, about50-fold, about 100-fold, or about 1000-fold. In certain embodiments, thereduction in the number or frequency of cancer stem cells is determinedby limiting dilution assay using an animal model. Additional examplesand guidance regarding the use of limiting dilution assays to determinea reduction in the number or frequency of cancer stem cells in a tumorcan be found, e.g., in International Publication Number WO 2008/042236;U.S. Patent Publication No. 2008/0064049; and U.S. Patent PublicationNo. 2008/0178305.

In certain embodiments, the VEGF/DLL4-binding agents are capable ofmodulating angiogenesis. In certain embodiments, the VEGF/DLL4-bindingagents are capable of modulating angiogenesis in vivo (e.g., in axenograft mouse model, and/or in a human having cancer). In certainembodiments, VEGF/DLL4-binding agents are capable of inhibitingangiogenesis. In certain embodiments, VEGF/DLL4-binding agents arecapable of promoting aberrant angiogenesis. In certain embodiments,VEGF/DLL4-binding agents are capable of inhibiting angiogenesis and/orpromoting aberrant angiogenesis, leading to unproductivevascularization.

In certain embodiments, the VEGF/DLL4-binding agents described hereinhave a circulating half-life in mice, cynomolgus monkeys, or humans ofat least about 2 hours, at least about 5 hours, at least about 10 hours,at least about 24 hours, at least about 3 days, at least about 1 week,or at least about 2 weeks. In certain embodiments, the VEGF/DLL4-bindingagent is an IgG (e.g., IgG1 or IgG2) antibody that has a circulatinghalf-life in mice, cynomolgus monkeys, or humans of at least about 2hours, at least about 5 hours, at least about 10 hours, at least about24 hours, at least about 3 days, at least about 1 week, or at leastabout 2 weeks. Methods of increasing (or decreasing) the half-life ofagents such as polypeptides and antibodies are known in the art. Forexample, known methods of increasing the circulating half-life of IgGantibodies include the introduction of mutations in the Fc region whichincrease the pH-dependent binding of the antibody to the neonatal Fcreceptor (FcRn) at pH 6.0 (see, e.g., U.S. Patent Publication Nos.2005/0276799, 2007/0148164, and 2007/0122403). Known methods ofincreasing the circulating half-life of antibody fragments lacking theFc region include such techniques as PEGylation.

In some embodiments, the VEGF/DLL4-binding agents are antibodies.Polyclonal antibodies can be prepared by any known method. In someembodiments, polyclonal antibodies are produced by immunizing an animal(e.g., a rabbit, rat, mouse, goat, donkey) with an antigen of interest(e.g., a purified peptide fragment, full-length recombinant protein, orfusion protein) by multiple subcutaneous or intraperitoneal injections.The antigen can be optionally conjugated to a carrier such as keyholelimpet hemocyanin (KLH) or serum albumin. The antigen (with or without acarrier protein) is diluted in sterile saline and usually combined withan adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form astable emulsion. After a sufficient period of time, polyclonalantibodies are recovered from the immunized animal, usually from bloodor ascites. The polyclonal antibodies can be purified from serum orascites according to standard methods in the art including, but notlimited to, affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In some embodiments, the VEGF/DLL4-binding agents are monoclonalantibodies. Monoclonal antibodies can be prepared using hybridomamethods known to one of skill in the art (see e.g., Kohler and Milstein,1975, Nature, 256:495-497). In some embodiments, using the hybridomamethod, a mouse, hamster, or other appropriate host animal, is immunizedas described above to elicit from lymphocytes the production ofantibodies that specifically bind the immunizing antigen. In someembodiments, lymphocytes can be immunized in vitro. In some embodiments,the immunizing antigen can be a human protein or a portion thereof. Insome embodiments, the immunizing antigen can be a mouse protein or aportion thereof.

Following immunization, lymphocytes are isolated and fused with asuitable myeloma cell line using, for example, polyethylene glycol. Thehybridoma cells are selected using specialized media as known in the artand unfused lymphocytes and myeloma cells do not survive the selectionprocess. Hybridomas that produce monoclonal antibodies directedspecifically against a chosen antigen may be identified by a variety ofmethods including, but not limited to, immunoprecipitation,immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS,ELISA, and radioimmunoassay). The hybridomas can be propagated either inin vitro culture using standard methods (J. W. Goding, 1996, MonoclonalAntibodies: Principles and Practice, 3^(rd) Edition, Academic Press, SanDiego, Calif.) or in vivo as ascites tumors in an animal. The monoclonalantibodies can be purified from the culture medium or ascites fluidaccording to standard methods in the art including, but not limited to,affinity chromatography, ion-exchange chromatography, gelelectrophoresis, and dialysis.

In certain embodiments, monoclonal antibodies can be made usingrecombinant DNA techniques as known to one skilled in the art. Thepolynucleotides encoding a monoclonal antibody are isolated from matureB-cells or hybridoma cells, such as by RT-PCR using oligonucleotideprimers that specifically amplify the genes encoding the heavy and lightchains of the antibody, and their sequence is determined using standardtechniques. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors which producethe monoclonal antibodies when transfected into host cells such as E.coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myelomacells that do not otherwise produce immunoglobulin proteins.

In certain other embodiments, recombinant monoclonal antibodies, orfragments thereof, can be isolated from phage display librariesexpressing variable domains or CDRs of a desired species (see e.g.,McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can be modified,for example, by using recombinant DNA technology to generate alternativeantibodies. In some embodiments, the constant domains of the light andheavy chains of, for example, a mouse monoclonal antibody can besubstituted for those regions of, for example, a human antibody togenerate a chimeric antibody, or for a non-immunoglobulin polypeptide togenerate a fusion antibody. In some embodiments, the constant regionsare truncated or removed to generate the desired antibody fragment of amonoclonal antibody. Site-directed or high-density mutagenesis of thevariable region can be used to optimize specificity, affinity, etc. of amonoclonal antibody.

In some embodiments, a monoclonal antibody against VEGF and/or DLL4 is ahumanized antibody. Typically, humanized antibodies are humanimmunoglobulins in which residues from the CDRs are replaced by residuesfrom a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster,etc.) that have the desired specificity, affinity, and/or bindingcapability using methods known to one skilled in the art. In someembodiments, the Fv framework region residues of a human immunoglobulinare replaced with the corresponding residues in an antibody from anon-human species that has the desired specificity, affinity, and/orbinding capability. In some embodiments, a humanized antibody can befurther modified by the substitution of additional residues either inthe Fv framework region and/or within the replaced non-human residues torefine and optimize antibody specificity, affinity, and/or capability.In general, a humanized antibody will comprise substantially all of atleast one, and typically two or three, variable domain regionscontaining all, or substantially all, of the CDRs that correspond to thenon-human immunoglobulin whereas all, or substantially all, of theframework regions are those of a human immunoglobulin consensussequence. In some embodiments, a humanized antibody can also comprise atleast a portion of an immunoglobulin constant region or domain (Fc),typically that of a human immunoglobulin. In certain embodiments, suchhumanized antibodies are used therapeutically because they may reduceantigenicity and HAMA (human anti-mouse antibody) responses whenadministered to a human subject. One skilled in the art would be able toobtain a functional humanized antibody with reduced immunogenicityfollowing known techniques (see e.g., U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; and 5,693,762).

In certain embodiments, the VEGF/DLL4-binding agent is a human antibody.Human antibodies can be directly prepared using various techniques knownin the art. In some embodiments, human antibodies may be generated fromimmortalized human B lymphocytes immunized in vitro or from lymphocytesisolated from an immunized individual. In either case, cells thatproduce an antibody directed against a target antigen can be generatedand isolated (see, e.g., Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77; Boemer et al., 1991, J. Immunol.,147:86-95; and U.S. Pat. Nos. 5,750,373; 5,567,610; and 5,229,275). Insome embodiments, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Alternatively, phage displaytechnology can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108;6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2008, J. Mol. Bio., 376:1182-1200. Once antibodies are identified,affinity maturation strategies known in the art, including but notlimited to, chain shuffling (Marks et al., 1992, Bio/Technology,10:779-783) and site-directed mutagenesis, may be employed to generatehigh affinity human antibodies.

In some embodiments, human antibodies can be made in transgenic micethat contain human immunoglobulin loci. Upon immunization these mice arecapable of producing the full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. This approach isdescribed in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies. Bispecificantibodies are capable of specifically recognizing and binding at leasttwo different antigens or epitopes. The different epitopes can either bewithin the same molecule (e.g., two epitopes on a single protein) or ondifferent molecules (e.g., one epitope on a protein and one epitope on asecond protein). In some embodiments, a bispecific antibody has enhancedpotency as compared to an individual antibody or to a combination ofmore than one antibody. In some embodiments, a bispecific antibody hasreduced toxicity as compared to an individual antibody or to acombination of more than one antibody. It is known to those of skill inthe art that any binding agent (e.g., antibody) may have uniquepharmacokinetics (PK) (e.g., circulating half-life). In someembodiments, a bispecific antibody has the ability to synchronize the PKof two active binding agents wherein the two individual binding agentshave different PK profiles. In some embodiments, a bispecific antibodyhas the ability to concentrate the actions of two binding agents (e.g.,antibodies) in a common area (e.g., a tumor and/or tumor environment).In some embodiments, a bispecific antibody has the ability toconcentrate the actions of two binding agents (e.g., antibodies) to acommon target (e.g., a tumor or a tumor cell). In some embodiments, abispecific antibody has the ability to target the actions of two bindingagents (e.g., antibodies) to more than one biological pathway orfunction.

In certain embodiments, the bispecific antibody specifically binds VEGFand a second target. In certain embodiments, the bispecific antibodyspecifically binds DLL4 and a second target. In certain embodiments, thebispecific antibody specifically binds VEGF and DLL4. In someembodiments, the bispecific antibody specifically binds human VEGF andhuman DLL4. In some embodiments, the bispecific antibody is a monoclonalhuman or a humanized antibody. In some embodiments, the bispecificantibody inhibits angiogenesis and reduces cancer stem cell number orfrequency. In some embodiments, the bispecific antibody inhibits bloodvessel growth and inhibits blood vessel maturation. In some embodiments,the bispecific antibody prevents endothelial hyperproliferation. In someembodiments, the bispecific antibody has decreased toxicity and/or sideeffects. In some embodiments, the bispecific antibody has decreasedtoxicity and/or side effects as compared to a mixture of the twoindividual antibodies or the antibodies as single agents. In someembodiments, the bispecific antibody has an increased therapeutic index.In some embodiments, the bispecific antibody has an increasedtherapeutic index as compared to a mixture of the two individualantibodies or the antibodies as single agents.

In some embodiments, the bispecific antibody can specifically recognizeand bind a first antigen target, (e.g., DLL4) as well as a secondantigen target, such as an effector molecule on a leukocyte (e.g., CD2,CD3, CD28, or B7) or a Fc receptor (e.g., CD64, CD32, or CD16) so as tofocus cellular defense mechanisms to the cell expressing the firstantigen target. In some embodiments, the bispecific antibodies can beused to direct cytotoxic agents to cells which express a particulartarget antigen. These antibodies possess an antigen-binding site (e.g.,to human DLL4) and a second site which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.

Techniques for making bispecific antibodies are known by those skilledin the art, see for example, Millstein et al., 1983, Nature,305:537-539; Brennan et al., 1985, Science, 229:81; Suresh et al., 1986,Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBO J.,10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225; Kostelnyet al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994, J.Immunol., 152:5368; U.S. Pat. No. 5,731,168; International PublicationNo. WO 2009/089004; and U.S. Patent Publication No. 2011/0123532. Insome embodiments, the bispecific antibodies comprise heavy chainconstant regions with modifications in the amino acids which are part ofthe interface between the two heavy chains. In some embodiments, thebispecific antibodies can be generated using a “knobs-into-holes”strategy (see. e.g., U.S. Pat. No. 5,731,168; Ridgway et. al., 1996,Prot. Engin., 9:617-621). At times the “knobs” and “holes” terminologyis replaced with the terms “protuberances” and “cavities”. In someembodiments, the bispecific antibodies may comprise variant hingeregions incapable of forming disulfide linkages between the heavy chains(see, e.g., WO 2006/028936). In some embodiments, the modifications maycomprise changes in amino acids that result in altered electrostaticinteractions. In some embodiments, the modifications may comprisechanges in amino acids that result in altered hydrophobic/hydrophilicinteractions.

Bispecific antibodies can be intact antibodies or antibody fragmentscomprising antigen-binding sites. Antibodies with more than twovalencies are also contemplated. For example, trispecific antibodies canbe prepared (Tutt et al., 1991, J. Immunol., 147:60). Thus, in certainembodiments the antibodies to VEGF and/or DLL4 are multispecific.

In certain embodiments, the antibodies (or other polypeptides) describedherein may be monospecific. In certain embodiments, each of the one ormore antigen-binding sites that an antibody contains is capable ofbinding (or binds) a homologous epitope on different proteins.

In certain embodiments, the VEGF/DLL4-binding agent is an antibodyfragment. Antibody fragments may have different functions orcapabilities than intact antibodies; for example, antibody fragments canhave increased tumor penetration. Various techniques are known for theproduction of antibody fragments including, but not limited to,proteolytic digestion of intact antibodies. In some embodiments,antibody fragments include a F(ab′)2 fragment produced by pepsindigestion of an antibody molecule. In some embodiments, antibodyfragments include a Fab fragment generated by reducing the disulfidebridges of an F(ab′)2 fragment. In other embodiments, antibody fragmentsinclude a Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent. In certain embodiments,antibody fragments are produced recombinantly. In some embodiments,antibody fragments include Fv or single chain Fv (scFv) fragments. Fab,Fv, and scFv antibody fragments can be expressed in and secreted from E.coli or other host cells, allowing for the production of large amountsof these fragments. In some embodiments, antibody fragments are isolatedfrom antibody phage libraries as discussed herein. For example, methodscan be used for the construction of Fab expression libraries (Huse etal., 1989, Science, 246:1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor VEGF and/or DLL4 or derivatives, fragments, analogs or homologsthereof. In some embodiments, antibody fragments are linear antibodyfragments. In certain embodiments, antibody fragments are monospecificor bispecific. In certain embodiments, the VEGF/DLL4-binding agent is ascFv. Various techniques can be used for the production of single-chainantibodies specific to VEGF or DLL4 (see, e.g., U.S. Pat. No.4,946,778).

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to alter (e.g., increase ordecrease) its serum half-life. This can be achieved, for example, byincorporation of a salvage receptor binding epitope into the antibodyfragment by mutation of the appropriate region in the antibody fragmentor by incorporating the epitope into a peptide tag that is then fused tothe antibody fragment at either end or in the middle (e.g., by DNA orpeptide synthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (see, e.g., U.S. Pat. No.4,676,980). It is also contemplated that the heteroconjugate antibodiescan be prepared in vitro using known methods in synthetic proteinchemistry, including those involving crosslinking agents. For example,immunotoxins can be constructed using a disulfide exchange reaction orby forming a thioether bond. Examples of suitable reagents for thispurpose include iminothiolate and methyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the target (i.e.,human VEGF or human DLL4). In this regard, the variable region maycomprise or be derived from any type of mammal that can be induced tomount a humoral response and generate immunoglobulins against thedesired antigen. As such, the variable region of the modified antibodiescan be, for example, of human, murine, non-human primate (e.g.cynomolgus monkeys, macaques, etc.) or rabbit origin. In someembodiments, both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments, the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencemodification and/or alteration. Although the CDRs may be derived from anantibody of the same class or even subclass as the antibody from whichthe framework regions are derived, it is envisaged that the CDRs may bederived from an antibody of different class and often from an antibodyfrom a different species. It may not be necessary to replace all of theCDRs with all of the CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are required tomaintain the activity of the antigen-binding site.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or increased serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. The modified antibodies disclosed hereinmay comprise alterations or modifications to one or more of the threeheavy chain constant domains (CH1, CH2 or CH3) and/or to the light chainconstant domain (CL). In some embodiments, one or more domains arepartially or entirely deleted from the constant regions of the modifiedantibodies. In some embodiments, the modified antibodies will comprisedomain deleted constructs or variants wherein the entire CH2 domain hasbeen removed (ACH2 constructs). In some embodiments, the omittedconstant region domain is replaced by a short amino acid spacer (e.g.,10 amino acid residues) that provides some of the molecular flexibilitytypically imparted by the absent constant region.

In some embodiments, the modified antibodies are engineered to fuse theCH3 domain directly to the hinge region of the antibody. In otherembodiments, a peptide spacer is inserted between the hinge region andthe modified CH2 and/or CH3 domains. For example, constructs may beexpressed wherein the CH2 domain has been deleted and the remaining CH3domain (modified or unmodified) is joined to the hinge region with a5-20 amino acid spacer. Such a spacer may be added to ensure that theregulatory elements of the constant domain remain free and accessible orthat the hinge region remains flexible. However, it should be noted thatamino acid spacers may, in some cases, prove to be immunogenic andelicit an unwanted immune response against the construct. Accordingly,in certain embodiments, any spacer added to the construct will berelatively non-immunogenic so as to maintain the desired biologicalqualities of the modified antibodies.

In some embodiments, the modified antibodies may have only a partialdeletion of a constant domain or substitution of a few or even a singleamino acid. For example, the mutation of a single amino acid in selectedareas of the CH2 domain may be enough to substantially reduce Fc bindingand thereby increase cancer cell localization and/or tumor penetration.Similarly, it may be desirable to simply delete the part of one or moreconstant region domains that control a specific effector function (e.g.complement C1q binding) to be modulated. Such partial deletions of theconstant regions may improve selected characteristics of the antibody(serum half-life) while leaving other desirable functions associatedwith the subject constant region domain intact. Moreover, as alluded toabove, the constant regions of the disclosed antibodies may be modifiedthrough the mutation or substitution of one or more amino acids thatenhances the profile of the resulting construct. In this respect it maybe possible to disrupt the activity provided by a conserved binding site(e.g., Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. In certain embodiments,the modified antibodies comprise the addition of one or more amino acidsto the constant region to enhance desirable characteristics such asdecreasing or increasing effector function or provide for more cytotoxinor carbohydrate attachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan antibody can bind a cell expressing a Fc receptor (FcR). There are anumber of Fc receptors which are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (called antibody-dependentcell cytotoxicity or ADCC), release of inflammatory mediators, placentaltransfer, and control of immunoglobulin production.

In certain embodiments, the modified antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, in some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating modifiedantibody thereby increasing cancer cell localization and/or tumorpenetration. In other embodiments, the constant region modificationsincrease the serum half-life of the antibody. In other embodiments, theconstant region modifications reduce the serum half-life of theantibody. In some embodiments, the constant region is modified toeliminate disulfide linkages or oligosaccharide moieties. Modificationsto the constant region in accordance with this invention may easily bemade using well known biochemical or molecular engineering techniquesknown to those of skill in the art.

In certain embodiments, a VEGF/DLL4-binding agent that is an antibodydoes not have one or more effector functions. For instance, in someembodiments, the antibody has no ADCC activity, and/or nocomplement-dependent cytotoxicity (CDC) activity. In certainembodiments, the antibody does not bind an Fc receptor, and/orcomplement factors. In certain embodiments, the antibody has no effectorfunction.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized, and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another amino acid within the same general class suchas, for example, one acidic amino acid with another acidic amino acid,one basic amino acid with another basic amino acid or one neutral aminoacid by another neutral amino acid. What is intended by a conservativeamino acid substitution is well known in the art and described herein.

Thus, the present invention provides methods for producing an antibodythat binds VEGF and/or DLL4, including bispecific antibodies thatspecifically bind both VEGF and DLL4. In some embodiments, the methodfor producing an antibody that binds VEGF and/or DLL4 comprises usinghybridoma techniques. In some embodiments, the method of generating anantibody that binds VEGF or DLL4 or a bispecific antibody that bindsVEGF and DLL4 comprises screening a human phage library. The presentinvention further provides methods of identifying an antibody that bindsVEGF and/or DLL4. In some embodiments, the antibody is identified byFACS screening for binding to VEGF or a portion thereof. In someembodiments, the antibody is identified by FACS screening for binding toDLL4 or a portion thereof. In some embodiments, the antibody isidentified by FACS screening for binding to both VEGF and DLL4 or aportion thereof. In some embodiments, the antibody is identified byscreening using ELISA for binding to VEGF. In some embodiments, theantibody is identified by screening using ELISA for binding to DLL4. Insome embodiments, the antibody is identified by screening using ELISAfor binding to VEGF and DLL4. In some embodiments, the antibody isidentified by FACS screening for blocking of binding of human VEGF to ahuman VEGF receptor. In some embodiments, the antibody is identified byFACS screening for blocking of binding of human DLL4 to a human Notchreceptor. In some embodiments, the antibody is identified by screeningfor inhibition or blocking of Notch signaling. In some embodiments, theantibody is identified by screening for inhibition or blocking of VEGFactivity (e.g., induction of HUVEC proliferation). In some embodiments,the antibody is identified by screening for modulation of angiogenesis.

In some embodiments, a method of generating an antibody to human VEGFcomprises immunizing a mammal with a polypeptide comprising amino acids27-232 of human VEGF. In some embodiments, a method of generating anantibody to human VEGF comprises immunizing a mammal with a polypeptidecomprising at least a portion of amino acids 27-232 of human VEGF. Insome embodiments, the method further comprises isolating antibodies orantibody-producing cells from the mammal. In some embodiments, a methodof generating a monoclonal antibody which binds VEGF comprises:immunizing a mammal with a polypeptide comprising at least a portion ofamino acids 27-232 of human VEGF, and isolating antibody-producing cellsfrom the immunized mammal. In some embodiments, the method furthercomprises fusing the antibody-producing cells with cells of a myelomacell line to form hybridoma cells. In some embodiments, the methodfurther comprises selecting a hybridoma cell expressing an antibody thatbinds VEGF. In certain embodiments, the mammal is a mouse. In someembodiments, the antibody is selected using a polypeptide comprising atleast a portion of amino acids 27-232 of human VEGF.

In some embodiments, a method of generating an antibody to human DLL4comprises immunizing a mammal with a polypeptide comprising amino acids27-529 of human DLL4. In some embodiments, a method of generating anantibody to human DLL4 comprises immunizing a mammal with a polypeptidecomprising at least a portion of amino acids 27-529 of human DLL4. Insome embodiments, a method of generating a monoclonal antibody whichbinds DLL4 comprises: immunizing a mammal with a polypeptide comprisingat least a portion of amino acids 27-529 of human DLL4, and isolatingantibody producing cells from the immunized mammal. In some embodiments,the method further comprises fusing the antibody-producing cells withcells of a myeloma cell line to form hybridoma cells. In someembodiments, the method further comprises selecting a hybridoma cellexpressing an antibody that binds DLL4. In certain embodiments, themammal is a mouse. In some embodiments, the antibody is selected using apolypeptide comprising at least a portion of amino acids 27-529 of humanDLL4.

In some embodiments, a method of generating an antibody to human VEGFcomprises screening an antibody-expressing library for antibodies thatbind human VEGF. In some embodiments, a method of generating an antibodyhuman DLL4 comprises screening an antibody-expressing library forantibodies that bind human DLL4. In some embodiments, a method ofgenerating an antibody to human VEGF and/or human DLL4 comprisesscreening an antibody-expressing library for bispecific antibodies thatbind human VEGF and human DLL4. In some embodiments, theantibody-expressing library is a phage library. In some embodiments, thescreening comprises panning. In some embodiments, theantibody-expressing library (e.g., a phage library) is screened using atleast a portion of amino acids 27-232 of human VEGF. In someembodiments, antibodies identified in the first screening, are screenedagain using at least a portion of amino acids 27-529 of human DLL4 toidentify a bispecific antibody that binds VEGF and DLL4. In someembodiments, the antibody-expressing library (e.g., a phage library) isscreened using at least a portion of amino acids 27-529 of human DLL4.In some embodiments, antibodies identified in the first screening, arescreened again using at least a portion of amino acids 27-232 of humanVEGF to identify a bispecific antibody that binds VEGF and DLL4. In someembodiments, the antibody identified in the screening is a VEGFantagonist. In some embodiments, the antibody identified in thescreening inhibits biological activities induced by VEGF. In someembodiments, the antibody identified in the screening is a DLL4antagonist. In some embodiments, the antibody identified in thescreening inhibits Notch signaling induced by DLL4. In some embodiments,the antibody identified in the screening binds both human VEGF and mouseVEGF. In some embodiments, the antibody identified in the screeningbinds both human DLL4 and mouse DLL4.

In certain embodiments, the antibodies described herein are isolated. Incertain embodiments, the antibodies described herein are substantiallypure.

In some embodiments of the present invention, the VEGF/DLL4-bindingagents are polypeptides. The polypeptides can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, that bind VEGF and/or DLL4. It will berecognized in the art that some amino acid sequences of the bindingagents described herein can be varied without significant effect on thestructure or function of the protein. Thus, the invention furtherincludes variations of the polypeptides which show substantial activityor which include regions of an antibody, or fragment thereof, againsthuman VEGF and/or DLL4. In some embodiments, amino acid sequencevariations of VEGF/DLL4-binding polypeptides include deletions,insertions, inversions, repeats, and/or other types of substitutions.

In some embodiments, the polypeptides described herein are isolated. Insome embodiments, the polypeptides described herein are substantiallypure.

The polypeptides, analogs and variants thereof, can be further modifiedto contain additional chemical moieties not normally part of thepolypeptide. The derivatized moieties can improve or otherwise modulatethe solubility, the biological half-life, and/or absorption of thepolypeptide. The moieties can also reduce or eliminate undesirable sideeffects of the polypeptides and variants. An overview for chemicalmoieties can be found in Remington: The Science and Practice ofPharmacy, 21^(st) Edition, 2005, University of the Sciences,Philadelphia, Pa.

The polypeptides described herein can be produced by any suitable methodknown in the art. Such methods range from direct protein synthesismethods to constructing a DNA sequence encoding polypeptide sequencesand expressing those sequences in a suitable host. In some embodiments,a DNA sequence is constructed using recombinant technology by isolatingor synthesizing a DNA sequence encoding a wild-type protein of interest.Optionally, the sequence can be mutagenized by site-specific mutagenesisto provide functional analogs thereof. See, e.g., Zoeller et al., 1984,PNAS, 81:5662-5066 and U.S. Pat. No. 4,588,585.

In some embodiments, a DNA sequence encoding a polypeptide of interestmay be constructed by chemical synthesis using an oligonucleotidesynthesizer. Oligonucleotides can be designed based on the amino acidsequence of the desired polypeptide and selecting those codons that arefavored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the polynucleotide sequences encoding a particular polypeptideof interest can be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe protein in a desired host. Proper assembly can be confirmed bynucleotide sequencing, restriction enzyme mapping, and/or expression ofa biologically active polypeptide in a suitable host. As is well-knownin the art, in order to obtain high expression levels of a transfectedgene in a host, the gene must be operatively linked to transcriptionaland translational expression control sequences that are functional inthe chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human VEGF and/or DLL4. For example, recombinant expressionvectors can be replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a polypeptide chain of aVEGF/DLL4-binding agent, such as an anti-VEGF antibody or an anti-DLL4antibody, or fragment thereof, operatively linked to suitabletranscriptional and/or translational regulatory elements derived frommammalian, microbial, viral, or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences. Regulatory elements can include an operator sequence tocontrol transcription. The ability to replicate in a host, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are “operatively linked” when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. In some embodiments,structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. In other embodiments, in situations where recombinantprotein is expressed without a leader or transport sequence, it caninclude an N-terminal methionine residue. This residue can optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

The choice of an expression control sequence and an expression vectordepends upon the choice of host. A wide variety of expressionhost/vector combinations can be employed. Useful expression vectors foreukaryotic hosts include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus, andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1,pBR322, pMB9, and their derivatives, and wider host range plasmids, suchas M13 and other filamentous single-stranded DNA phages.

The VEGF/DLL4-binding agents (e.g., polypeptides) of the presentinvention can be expressed from one or more vectors. For example, insome embodiments, one heavy chain polypeptide is expressed by onevector, a second heavy chain polypeptide is expressed by a second vectorand a light chain polypeptide is expressed by a third vector. In someembodiments, a first heavy chain polypeptide and a light chainpolypeptide is expressed by one vector and a second heavy chainpolypeptide is expressed by a second vector. In some embodiments, twoheavy chain polypeptides are expressed by one vector and a light chainpolypeptide is expressed by a second vector. In some embodiments, threepolypeptides are expressed from one vector. Thus, in some embodiments, afirst heavy chain polypeptide, a second heavy chain polypeptide, and alight chain polypeptide are expressed by a single vector.

Suitable host cells for expression of a VEGF/DLL4-binding polypeptide orantibody (or a VEGF or DLL4 protein to use as an antigen) includeprokaryotes, yeast cells, insect cells, or higher eukaryotic cells underthe control of appropriate promoters. Prokaryotes include gram-negativeor gram-positive organisms, for example E. coli or Bacillus. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems may also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described in Pouwels etal., 1985, Cloning Vectors: A Laboratory Manual, Elsevier, New York,N.Y. Additional information regarding methods of protein production,including antibody production, can be found, e.g., in U.S. PatentPublication No. 2008/0187954; U.S. Pat. Nos. 6,413,746; 6,660,501; andInternational Patent Publication No. WO 04/009823.

Various mammalian or insect cell culture systems may be used to expressrecombinant polypeptides. Expression of recombinant proteins inmammalian cells may be desirable because these proteins are generallycorrectly folded, appropriately modified, and biologically functional.Examples of suitable mammalian host cell lines include, but are notlimited to, COS-7 (monkey kidney-derived), L-929 (murinefibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murinefibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (humancervical cancer-derived), BHK (hamster kidney fibroblast-derived),HEK-293 (human embryonic kidney-derived) cell lines and variants ofthese cell lines. Mammalian expression vectors can comprisenon-transcribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Expression of recombinant proteins in baculovirus also offersa robust method for producing correctly folded and biologicallyfunctional proteins. Baculovirus systems for production of heterologousproteins in insect cells are well-known to those of skill in the art(see, e.g., Luckow and Summers, 1988, Bio/Technology, 6:47).

Thus, the present invention provides cells comprising theVEGF/DLL4-binding agents described herein. In some embodiments, thecells produce the VEGF/DLL4-binding agents described herein. In certainembodiments, the cells produce an antibody. In some embodiments, thecells produce a VEGF-binding agent, such as an anti-VEGF antibody. Insome embodiments, the cells produce a bispecific antibody that bindsVEGF. In some embodiments, the cells produce a DLL4-binding agent, suchas an anti-DLL4 antibody. In some embodiments, the cells produce abispecific antibody that binds DLL4. In certain embodiments, the cellsproduce a bispecific VEGF/DLL4-binding agent, such as a bispecificantibody that binds VEGF and DLL4. In certain embodiments, the cellsproduce antibody 219R45. In certain embodiments, the cells produceantibody 21R79. In certain embodiments, the cells produce antibody21R75. In certain embodiments, the cells produce antibody 21R83. Incertain embodiments, the cells produce a bispecific antibody whichcomprises an antigen-binding site from antibody 219R45. In certainembodiments, the cells produce a bispecific antibody which comprises anantigen-binding site from antibody 21R79. In certain embodiments, thecells produce a bispecific antibody which comprises an antigen-bindingsite from antibody 21R75. In certain embodiments, the cells produce abispecific antibody which comprises an antigen-binding site fromantibody 21R83. In certain embodiments, the cells produce a bispecificantibody which comprises an antigen-binding site from antibody 219R45and an antigen-binding site from antibody 21R79. In certain embodiments,the cells produce a bispecific antibody which comprises anantigen-binding site from antibody 219R45 and an antigen-binding sitefrom antibody 21M18. In certain embodiments, the cells produce abispecific antibody which comprises an antigen-binding site fromantibody 219R45 and an antigen-binding site from antibody 21R75. Incertain embodiments, the cells produce a bispecific antibody whichcomprises an antigen-binding site from antibody 219R45 and anantigen-binding site from antibody 21R83. In certain embodiments, thecells produce the bispecific antibody 219R45-MB-21M18. In certainembodiments, the cells produce the bispecific antibody 219R45-MB-21R79.In certain embodiments, the cells produce the bispecific antibody219R45-MB-21R75. In certain embodiments, the cells produce thebispecific antibody 219R45-MB-21R83.

The proteins produced by a transformed host can be purified according toany suitable method. Standard methods include chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. Affinity tags such as hexa-histidine, maltose bindingdomain, influenza coat sequence, and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Affinity chromatography used for purifyingimmunoglobulins can include Protein A, Protein G, and Protein Lchromatography. Isolated proteins can be physically characterized usingsuch techniques as proteolysis, size exclusion chromatography (SEC),mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectricfocusing (IEF), high performance liquid chromatography (HPLC), and x-raycrystallography. The purity of isolated proteins can be determined usingtechniques known to those of skill in the art, including but not limitedto, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillaryisoelectric focusing (cIEF).

In some embodiments, supernatants from expression systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite media can be employed, including but not limited to,ceramic hydroxyapatite (CHT). In certain embodiments, one or morereverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a recombinant protein (e.g., aVEGF/DLL4-binding agent). Some or all of the foregoing purificationsteps, in various combinations, can be employed to provide a homogeneousrecombinant protein.

In some embodiments, heterodimeric proteins such as bispecificantibodies are purified according the any of the methods describedherein. In some embodiments, anti-VEGF/anti-DLL4 bispecific antibodiesare isolated and/or purified using at least one chromatography step. Insome embodiments, the at least one chromatography step comprisesaffinity chromatography. In some embodiments, the at least onechromatography step further comprises anion exchange chromatography. Insome embodiments, the isolated and/or purified antibody productcomprises at least 90% heterodimeric antibody. In some embodiments, theisolated and/or purified antibody product comprises at least 95%, 96%,97%, 98% or 99% heterodimeric antibody. In some embodiments, theisolated and/or purified antibody product comprises about 100%heterodimeric antibody.

In some embodiments, recombinant protein produced in bacterial culturecan be isolated, for example, by initial extraction from cell pellets,followed by one or more concentration, salting-out, aqueous ionexchange, or size exclusion chromatography steps. HPLC can be employedfor final purification steps. Microbial cells employed in expression ofa recombinant protein can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNos. 2008/0312425; 2008/0177048; and 2009/0187005.

In certain embodiments, the VEGF/DLL4-binding agent is a polypeptidethat is not an antibody. A variety of methods for identifying andproducing non-antibody polypeptides that bind with high affinity to aprotein target are known in the art. See, e.g., Skerra, 2007, Curr.Opin. Biotechnol., 18:295-304; Hosse et al., 2006, Protein Science,15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol., 17:653-658;Nygren, 2008, FEBS J., 275:2668-76; and Skerra, 2008, FEBS J.,275:2677-83. In certain embodiments, phage or mammalian cell displaytechnology may be used to produce and/or identify a VEGF/DLL4-bindingpolypeptide that is not an antibody. In certain embodiments, thepolypeptide comprises a protein scaffold of a type selected from thegroup consisting of protein A, protein G, a lipocalin, a fibronectindomain, an ankyrin consensus repeat domain, and thioredoxin.

In certain embodiments, the VEGF/DLL4-binding agents or antibodies canbe used in any one of a number of conjugated (i.e. an immunoconjugate orradioconjugate) or non-conjugated forms. In certain embodiments, theantibodies can be used in a non-conjugated form to harness the subject'snatural defense mechanisms including complement-dependent cytotoxicityand antibody-dependent cellular toxicity to eliminate malignant orcancer cells.

In some embodiments, the VEGF/DLL4-binding agent (e.g., an antibody orpolypeptide) is conjugated to a cytotoxic agent. In some embodiments,the cytotoxic agent is a chemotherapeutic agent including, but notlimited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents. In someembodiments, the cytotoxic agent is an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof,including, but not limited to, diphtheria A chain, non-binding activefragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. In some embodiments, the cytotoxicagent is a radioisotope to produce a radioconjugate or a radioconjugatedantibody. A variety of radionuclides are available for the production ofradioconjugated antibodies including, but not limited to, ⁹⁰Y, ¹²⁵I,¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re and ²¹²Bi. Conjugates of an antibody and one or more smallmolecule toxins, such as calicheamicins, maytansinoids, trichothecenes,and CC1065, and the derivatives of these toxins that have toxinactivity, can also be used. Conjugates of an antibody and cytotoxicagent can be made using a variety of bifunctional protein-couplingagents including, but not limited to,N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide (or a fragment of apolypeptide) that specifically binds VEGF, DLL4, both VEGF and DLL4. Theterm “polynucleotides that encode a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptide,as well as a polynucleotide which includes additional coding and/ornon-coding sequences. For example, in some embodiments, the inventionprovides a polynucleotide comprising a polynucleotide sequence thatencodes an antibody to human VEGF or encodes a fragment of such anantibody (e.g., a fragment comprising the antigen-binding site). In someembodiments, the invention provides a polynucleotide comprising apolynucleotide sequence that encodes an antibody to human DLL4 orencodes a fragment of such an antibody (e.g., a fragment comprising theantigen-binding site). The polynucleotides of the invention can be inthe form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA,and synthetic DNA; and can be double-stranded or single-stranded, and ifsingle-stranded can be the coding strand or non-coding (anti-sense)strand.

In certain embodiments, the polynucleotide comprises a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:62,SEQ ID NO:63, and SEQ ID NO:64. In certain embodiments, thepolynucleotide comprises a polynucleotide encoding a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:62, and SEQ ID NO: 64. In some embodiments, thepolynucleotide comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78.

In certain embodiments, the polynucleotide comprises a polynucleotidehaving a nucleotide sequence at least about 80% identical, at leastabout 85% identical, at least about 90% identical, at least about 95%identical, and in some embodiments, at least about 96%, 97%, 98% or 99%identical to a polynucleotide comprising a sequence selected from thegroup consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:66, SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQID NO:78. In certain embodiments, the polynucleotide comprises apolynucleotide having a nucleotide sequence at least about 80%identical, at least about 85% identical, at least about 90% identical,at least about 95% identical, and in some embodiments, at least about96%, 97%, 98% or 99% identical to a polynucleotide comprising a sequenceselected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, and SEQ ID NO:78. Also provided is a polynucleotidethat comprises a polynucleotide that hybridizes to SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, and SEQ ID NO:78. In certain embodiments, the hybridization isunder conditions of high stringency.

In certain embodiments, the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g., a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments, the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g., COS-7cells) is used. In some embodiments, the marker sequence is a FLAG-tag,a peptide of sequence DYKDDDDK (SEQ ID NO:45) which can be used inconjunction with other affinity tags.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs,and/or derivatives.

In certain embodiments, the present invention provides polynucleotidescomprising polynucleotides having a nucleotide sequence at least about80% identical, at least about 85% identical, at least about 90%identical, at least about 95% identical, and in some embodiments, atleast about 96%, 97%, 98% or 99% identical to a polynucleotide encodinga polypeptide comprising a VEGF/DLL4-binding agent (e.g., an antibody),or fragment thereof, described herein.

As used herein, the phrase a polynucleotide having a nucleotide sequenceat least, for example, 95% “identical” to a reference nucleotidesequence is intended to mean that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence can include up to five point mutations per each100 nucleotides of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence can be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence can be inserted into the referencesequence. These mutations of the reference sequence can occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, apolynucleotide variant contains alterations which produce silentsubstitutions, additions, or deletions, but does not alter theproperties or activities of the encoded polypeptide. In someembodiments, a polynucleotide variant comprises silent substitutionsthat results in no change to the amino acid sequence of the polypeptide(due to the degeneracy of the genetic code). Polynucleotide variants canbe produced for a variety of reasons, for example, to optimize codonexpression for a particular host (i.e., change codons in the human mRNAto those preferred by a bacterial host such as E. coli). In someembodiments, a polynucleotide variant comprises at least one silentmutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate oralter expression (or expression levels) of the encoded polypeptide. Insome embodiments, a polynucleotide variant is produced to increaseexpression of the encoded polypeptide. In some embodiments, apolynucleotide variant is produced to decrease expression of the encodedpolypeptide. In some embodiments, a polynucleotide variant has increasedexpression of the encoded polypeptide as compared to a parentalpolynucleotide sequence. In some embodiments, a polynucleotide varianthas decreased expression of the encoded polypeptide as compared to aparental polynucleotide sequence.

In some embodiments, at least one polynucleotide variant is produced(without changing the amino acid sequence of the encoded polypeptide) toincrease production of a heteromultimeric molecule. In some embodiments,at least one polynucleotide variant is produced (without changing theamino acid sequence of the encoded polypeptide) to increase productionof a bispecific antibody.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

Vectors and cells comprising the polynucleotides described herein arealso provided. In some embodiments, an expression vector comprises apolynucleotide molecule. In some embodiments, a host cell comprises anexpression vector comprising the polynucleotide molecule. In someembodiments, a host cell comprises a polynucleotide molecule.

IV. Methods of Use and Pharmaceutical Compositions

The -binding agents (including polypeptides and antibodies) of theinvention that bind (e.g., specifically bind) VEGF and/or DLL4 areuseful in a variety of applications including, but not limited to,therapeutic treatment methods, such as the treatment of cancer. Incertain embodiments, the agents are useful for inhibiting VEGF activity,inhibiting DLL4-induced Notch signaling, inhibiting tumor growth,reducing tumor volume, reducing the frequency of cancer stem cells in atumor, reducing the tumorigenicity of a tumor, modulating angiogenesis,and/or inhibiting angiogenesis. The methods of use may be in vitro, exvivo, or in vivo. In certain embodiments, a VEGF/DLL4-binding agent isan antagonist of human VEGF. In certain embodiments, a VEGF/DLL4-bindingagent is an antagonist of human DLL4. In certain embodiments, aVEGF/DLL4-binding agent is an antagonist of both VEGF and DLL4.

In certain embodiments, the VEGF/DLL4-binding agents are used in thetreatment of a disease associated with angiogenesis, i.e. increasedangiogenesis and/or aberrant angiogenesis. In certain embodiments, thedisease is a disease dependent upon angiogenesis. In certainembodiments, the VEGF/DLL4-binding agents are used in the treatment ofdisorders characterized by increased levels of stem cells and/orprogenitor cells.

The present invention provides methods for inhibiting growth of a tumorusing the VEGF/DLL4-binding agents or antibodies described herein. Incertain embodiments, the method of inhibiting growth of a tumorcomprises contacting a tumor cell with a VEGF/DLL4-binding agent (e.g.,antibody) in vitro. For example, an immortalized cell line or a cancercell line is cultured in medium to which is added an anti-VEGF antibody,an anti-DLL4 antibody, or an anti-VEGF/anti-DLL4 bispecific antibody toinhibit tumor cell growth. In some embodiments, tumor cells are isolatedfrom a patient sample such as, for example, a tissue biopsy, pleuraleffusion, or blood sample and cultured in medium to which is added aVEGF/DLL4-binding agent to inhibit tumor cell growth.

In some embodiments, the method of inhibiting growth of a tumorcomprises contacting a tumor or tumor cells with a VEGF/DLL4-bindingagent (e.g., antibody) in vivo. In certain embodiments, contacting atumor or tumor cell with a VEGF/DLL4-binding agent is undertaken in ananimal model. For example, an anti-VEGF antibody, an anti-DLL4 antibody,or an anti-VEGF/anti-DLL4 bispecific antibody may be administered to animmunocompromised host animal (e.g., NOD/SCID mice) which has a tumorxenograft. In some embodiments, tumor cells and/or cancer stem cells areisolated from a patient sample such as, for example, a tissue biopsy,pleural effusion, or blood sample and injected into an immunocompromisedhost animal (e.g., NOD/SCID mice) that is then administered aVEGF/DLL4-binding agent to inhibit tumor cell growth. In someembodiments, the VEGF/DLL4-binding agent is administered at the sametime or shortly after introduction of tumorigenic cells into the animalto prevent tumor growth (“preventative model”). In some embodiments, theVEGF/DLL4-binding agent is administered as a therapeutic after tumorshave grown to a specified size (“therapeutic model”). In certainembodiments, the VEGF/DLL4-binding agent is a bispecific antibody thatspecifically binds human VEGF and human DLL4.

In certain embodiments, the method of inhibiting growth of a tumorcomprises administering to a subject a therapeutically effective amountof a VEGF/DLL4-binding agent. In certain embodiments, the subject is ahuman. In certain embodiments, the subject has a tumor or has had atumor which was removed. In certain embodiments, the tumor comprisescancer stem cells. In certain embodiments, the frequency of cancer stemcells in the tumor is reduced by administration of the VEGF/DLL4-bindingagent. The invention also provides a method of reducing the frequency ofcancer stem cells in a tumor, comprising contacting the tumor with aneffective amount of a VEGF/DLL4-binding agent (e.g., ananti-VEGF/anti-DLL4 bispecific antibody). In some embodiments, a methodof reducing the frequency of cancer stem cells in a tumor in a subject,comprises administering to the subject a therapeutically effectiveamount of a VEGF/DLL4-binding agent.

In some embodiments, the tumor is a solid tumor. In certain embodiments,the tumor is a tumor selected from the group consisting of colorectaltumor, colon tumor, pancreatic tumor, lung tumor, ovarian tumor, livertumor, breast tumor, kidney tumor, prostate tumor, gastrointestinaltumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and headand neck tumor. In certain embodiments, the tumor is a colorectal tumoror a colon tumor. In certain embodiments, the tumor is an ovarian tumor.In some embodiments, the tumor is a lung tumor. In certain embodiments,the tumor is a pancreatic tumor. In certain embodiments, the tumor is abreast tumor.

The present invention further provides methods for treating cancercomprising administering a therapeutically effective amount of aVEGF/DLL4-binding agent to a subject. In some embodiments, theVEGF/DLL4-binding agent binds VEGF, and inhibits or reduces growth ofthe cancer. In some embodiments, the VEGF/DLL4-binding agent binds DLL4,and inhibits or reduces growth of the cancer. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody that binds VEGF andDLL4, and inhibits or reduces growth of the cancer. In some embodiments,the VEGF/DLL4-binding agent binds VEGF, interferes with VEGF/VEGFreceptor interactions, and inhibits or reduces growth of the cancer. Insome embodiments, the VEGF/DLL4-binding agent binds DLL4, interfereswith DLL4/Notch interactions, and inhibits or reduces growth of thecancer. In some embodiments, the VEGF/DLL4-binding agent binds both VEGFand DLL4, interferes with VEGF/VEGF receptor interactions and withDLL4/Notch interactions, and inhibits or reduces growth of the cancer.In some embodiments, the VEGF/DLL4-binding agent binds DLL4, and reducesthe frequency of cancer stem cells in the cancer.

The present invention provides methods of treating cancer comprisingadministering a therapeutically effective amount of a VEGF/DLL4-bindingagent to a subject (e.g., a subject in need of treatment). In certainembodiments, the subject is a human. In certain embodiments, the subjecthas a cancerous tumor. In certain embodiments, the subject has had atumor removed.

The subject's cancer/tumor, may, in some embodiments, be refractory tocertain treatment(s). As a non-limiting example, the subject's cancer(or tumor) may be chemorefractory. In certain embodiments, the subject'scancer may be resistant to anti-VEGF therapy or anti-DLL4 therapy, orboth.

In certain embodiments, the cancer is a cancer selected from the groupconsisting of colorectal cancer, pancreatic cancer, lung cancer, ovariancancer, liver cancer, breast cancer, kidney cancer, prostate cancer,gastrointestinal cancer, melanoma, cervical cancer, bladder cancer,glioblastoma, and head and neck cancer. In certain embodiments, thecancer is ovarian cancer. In certain embodiments, the cancer iscolorectal cancer or colon cancer. In certain embodiments, the cancer ispancreatic cancer. In certain embodiments, the cancer is breast cancer.In certain embodiments, the cancer is prostate cancer. In certainembodiments, the cancer is lung cancer. In some embodiments, the canceris a hematologic cancer such as leukemia or lymphoma. In someembodiments, the leukemia or lymphoma is a B-cell leukemia or lymphoma.In some embodiments, the leukemia or lymphoma is a T-cell leukemia orlymphoma. In some embodiments the hematologic cancer is acutemyelogenous leukemia, Hodgkin lymphoma, non-Hodgkins's lymphoma, acutelymphocytic leukemia, hairy cell leukemia, chronic lymphocytic leukemia,multiple myeloma, cutaneous T-cell lymphoma, or T-cell acutelymphoblastic leukemia.

The invention also provides methods of treating a disease or disorder ina subject, wherein the disease or disorder is associated withangiogenesis. In some embodiments, the disease or disorder is associatedwith aberrant angiogenesis. In some embodiments, the disease or disorderis associated with increased angiogenesis. Thus, the present inventionprovides methods for modulating angiogenesis in a subject, comprisingadministering to the subject a therapeutically effective amount of anyof the VEGF/DLL4-binding agents described herein. In some embodiments,the VEGF/DLL4-binding agent is an antibody that binds human VEGF. Insome embodiments, the VEGF/DLL4-binding agent is an antibody that bindshuman DLL4. In some embodiments, the VEGF/DLL4-binding agent is abispecific antibody that binds human VEGF. In some embodiments, theVEGF/DLL4-binding agent is a bispecific antibody that binds human DLL4.In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody that binds human VEGF and human DLL4.

Methods of treating a disease or disorder in a subject, wherein thedisease or disorder is characterized by an increased level of stem cellsand/or progenitor cells are further provided. In some embodiments, thetreatment methods comprise administering a therapeutically effectiveamount of a VEGF/DLL4-binding agent, polypeptide, or antibody to thesubject.

In certain embodiments of any of the methods described herein, theVEGF/DLL4-binding agent is a bispecific antibody that specifically bindshuman VEGF and human DLL4. In some embodiments, the bispecific antibodycomprises a first antigen-binding site that specifically binds humanVEGF and a second antigen-binding site that specifically binds humanDLL4, wherein the first antigen-binding site comprises a heavy chainCDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2 comprisingDINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3 comprisingHYDDKYYPLMDY (SEQ ID NO:19), and the second antigen-binding sitecomprises a heavy chain CDR1 comprising TAYYIH (SEQ ID NO:13), a heavychain CDR2 comprising YIANYNRATNYNQKFKG (SEQ ID NO:14),YISSYNGATNYNQKFKG (SEQ ID NO:15), YIAGYKDATNYNQKFKG (SEQ ID NO:59), orYISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIANYNRATNYNQKFKG (SEQ ID NO:14), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYISSYNGATNYNQKFKG (SEQ ID NO:15), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and second antigen-binding site which comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYIAGYKDATNYNQKFKG (SEQ ID NO:59), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22). In some embodiments, the bispecific antibody comprises a firstantigen-binding site that specifically binds human VEGF and a secondantigen-binding site that specifically binds human DLL4, wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19),and the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), a heavy chain CDR2 comprisingYISNYNRATNYNQKFKG (SEQ ID NO:65), and a heavy chain CDR3 comprisingRDYDYDVGMDY (SEQ ID NO:16); and wherein both the first and secondantigen-binding sites comprise a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and a light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).

In certain embodiments of any of the methods described herein, theVEGF/DLL4 bispecific antibody comprises a first heavy chain variableregion having at least about 80% sequence identity to SEQ ID NO:11, asecond heavy chain variable region having at least about 80% sequenceidentity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:58, or SEQ ID NO:64,and a first and a light chain variable region having at least 80%sequence identity to SEQ ID NO:12. In some embodiments, the VEGF/DLL4bispecific antibody comprises a first heavy chain variable region havingat least about 80% sequence identity to SEQ ID NO:11, a second heavychain variable region having at least about 80% sequence identity to SEQID NO:9, and a first and a second light chain variable region having atleast 80% sequence identity to SEQ ID NO:12. In some embodiments, theVEGF/DLL4 bispecific antibody comprises a first heavy chain variableregion having at least about 80% sequence identity to SEQ ID NO:11, asecond heavy chain variable region having at least about 80% sequenceidentity to SEQ ID NO:10, and a first and a second light chain variableregion having at least 80% sequence identity to SEQ ID NO:12. In someembodiments, the VEGF/DLL4 bispecific antibody comprises a first heavychain variable region having at least about 80% sequence identity to SEQID NO:11, a second heavy chain variable region having at least about 80%sequence identity to SEQ ID NO:58, and a first and a second light chainvariable region having at least 80% sequence identity to SEQ ID NO:12.In some embodiments, the VEGF/DLL4 bispecific antibody comprises a firstheavy chain variable region having at least about 80% sequence identityto SEQ ID NO:11, a second heavy chain variable region having at leastabout 80% sequence identity to SEQ ID NO:64, and a first and a secondlight chain variable region having at least 80% sequence identity to SEQID NO:12.

In some embodiments of any of the methods described herein, theVEGF/DLL4-binding agent is an antibody. In some embodiments, theVEGF/DLL4-binding agent is an anti-VEGF antibody. In some embodiments,the anti-VEGF antibody is antibody 219R45. In some embodiments, theVEGF/DLL4-binding agent is an anti-DLL4 antibody. In some embodiments,the anti-DLL4 antibody is antibody 21R79. In some embodiments, theanti-DLL4 antibody is antibody 21R75. In some embodiments, the anti-DLL4antibody is antibody 21R83. In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody comprising an antigen-binding site fromantibody 219R45. In some embodiments, the VEGF/DLL4-binding agent is abispecific antibody comprising an antigen-binding site from antibody21R79. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising an antigen-binding site from antibody 21R75. In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibodycomprising an antigen-binding site from antibody 21R83. In someembodiments, the VEGF/DLL4-binding agent is a bispecific antibodycomprising a first antigen-binding site from antibody 219R45 and asecond antigen-binding site from antibody 21R79. In some embodiments,the VEGF/DLL4-binding agent is a bispecific antibody comprising a firstantigen-binding site from antibody 219R45 and a second antigen-bindingsite from antibody 21M18. In some embodiments, the VEGF/DLL4-bindingagent is a bispecific antibody comprising a first antigen-binding sitefrom antibody 219R45 and a second antigen-binding site from antibody21R75. In some embodiments, the VEGF/DLL4-binding agent is a bispecificantibody comprising a first antigen-binding site from antibody 219R45and a second antigen-binding site from antibody 21R83. In someembodiments, the VEGF/DLL4-binding agent is the bispecific antibody219R45-MB-21M18. In some embodiments, the VEGF/DLL4-binding agent is thebispecific antibody 219R45-MB-21R79. In some embodiments, theVEGF/DLL4-binding agent is the bispecific antibody 219R45-MB-21R75. Insome embodiments, the VEGF/DLL4-binding agent is the bispecific antibody219R45-MB-21R83.

The present invention further provides pharmaceutical compositionscomprising the binding agents described herein. In certain embodiments,the pharmaceutical compositions further comprise a pharmaceuticallyacceptable vehicle. These pharmaceutical compositions find use ininhibiting tumor growth and/or treating cancer in a subject (e.g., ahuman patient).

In certain embodiments, the invention provides pharmaceuticalcompositions comprising bispecific antibodies, wherein at least about90%, at least about 95%, at least about 98%, at least about 99% of theantibodies in the composition are bispecific antibodies or heterodimericantibodies. In certain embodiments, the bispecific antibodies are IgG(e.g., IgG2 or IgG1) antibodies. In certain embodiments, less than about10%, less than about 5%, less than about 2% or less than about 1% of thetotal antibodies in the compositions are monospecific antibodies orhomodimeric antibodies. In certain embodiments, the antibodies in thecomposition are at least about 98% heterodimeric.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g., a carrier or excipient).Suitable pharmaceutically acceptable vehicles include, but are notlimited to, non-toxic buffers such as phosphate, citrate, and otherorganic acids; salts such as sodium chloride; antioxidants includingascorbic acid and methionine; preservatives such asoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride, benzethonium chloride, phenol, butyl or benzylalcohol, alkyl parabens, such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weightpolypeptides (e.g., less than about 10 amino acid residues); proteinssuch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosaccharides, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes suchas Zn-protein complexes; and non-ionic surfactants such as TWEEN orpolyethylene glycol (PEG). (Remington: The Science and Practice ofPharmacy, 21st Edition, 2005, University of the Sciences, Philadelphia,Pa.).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical by epidermal or transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids, and powders; pulmonary by inhalation or insufflation of powdersor aerosols, including by nebulizer, intratracheal, and intranasal;oral; or parenteral including intravenous, intraarterial, intratumoral,subcutaneous, intraperitoneal, intramuscular (e.g., injection orinfusion), or intracranial (e.g., intrathecal or intraventricular).

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, orsuppositories. In solid compositions such as tablets the principalactive ingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, anddiluents (e.g., water). These can be used to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a non-toxic pharmaceutically acceptable saltthereof. The solid preformulation composition is then subdivided intounit dosage forms of a type described above. The tablets, pills, etc. ofthe formulation or composition can be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner composition covered byan outer component. Furthermore, the two components can be separated byan enteric layer that serves to resist disintegration and permits theinner component to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials include a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The VEGF/DLL4-binding agents or antibodies described herein can also beentrapped in microcapsules. Such microcapsules are prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions asdescribed in Remington: The Science and Practice of Pharmacy, 21stEdition, 2005, University of the Sciences in Philadelphia, Pa.

In certain embodiments, pharmaceutical formulations include aVEGF/DLL4-binding agent (e.g., an antibody) of the present inventioncomplexed with liposomes. Methods to produce liposomes are known tothose of skill in the art. For example, some liposomes can be generatedby reverse phase evaporation with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes can be extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

In certain embodiments, sustained-release preparations can be produced.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing aVEGF/DLL4-binding agent (e.g., an antibody), where the matrices are inthe form of shaped articles (e.g., films or microcapsules). Additionalexamples of sustained-release matrices include polyesters, hydrogelssuch as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol),polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT™ (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

In certain embodiments, in addition to administering a VEGF/DLL4-bindingagent (e.g., an antibody), the method or treatment further comprisesadministering at least one additional therapeutic agent. An additionaltherapeutic agent can be administered prior to, concurrently with,and/or subsequently to, administration of the VEGF/DLL4-binding agent.Pharmaceutical compositions comprising a VEGF/DLL4-binding agent and theadditional therapeutic agent(s) are also provided. In some embodiments,the at least one additional therapeutic agent comprises 1, 2, 3, or moreadditional therapeutic agents.

Combination therapy with at least two therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of at least one of the agents. Combination therapy may decreasethe likelihood that resistant cancer cells will develop. In someembodiments, combination therapy comprises a therapeutic agent thatprimarily affects (e.g., inhibits or kills) non-tumorigenic cells and atherapeutic agent that primarily affects (e.g., inhibits or kills)tumorigenic CSCs.

Useful classes of therapeutic agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or thelike. In certain embodiments, the second therapeutic agent is analkylating agent, an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor. In some embodiments, the secondtherapeutic agent is a platinum complex such as carboplatin orcisplatin. In some embodiments, the additional therapeutic agent is aplatinum complex in combination with a taxane.

Therapeutic agents that may be administered in combination with theVEGF/DLL4-binding agents include chemotherapeutic agents. Thus, in someembodiments, the method or treatment involves the administration of ananti-VEGF-binding agent or antibody of the present invention incombination with a chemotherapeutic agent or cocktail of multipledifferent chemotherapeutic agents. In some embodiments, the method ortreatment involves the administration of an anti-DLL4-binding agent orantibody of the present invention in combination with a chemotherapeuticagent or cocktail of multiple different chemotherapeutic agents. In someembodiments, the method or treatment involves the administration of abispecific antibody of the present invention that binds VEGF and DLL4 incombination with a chemotherapeutic agent or cocktail of multipledifferent chemotherapeutic agents.

Chemotherapeutic agents useful in the instant invention include, but arenot limited to, alkylating agents such as thiotepa and cyclophosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel(TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including, for example, tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, the secondtherapeutic agent is cisplatin. In certain embodiments, the secondtherapeutic agent is carboplatin. In certain embodiments, the secondtherapeutic agent is paclitaxel.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapeutic agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese. In certain embodiments, the second therapeutic agent isirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the second therapeutic agent isgemcitabine.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain embodiments, the agentis paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel(ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor ofa mitotic kinase such as Aurora A or Plk1. In certain embodiments, wherethe chemotherapeutic agent administered in combination with aVEGF/DLL4-binding agent is an anti-mitotic agent, the cancer or tumorbeing treated is breast cancer or a breast tumor.

In some embodiments, a second therapeutic agent comprises an agent suchas a small molecule. For example, treatment can involve the combinedadministration of a VEGF/DLL4-binding agent (e.g. an antibody) of thepresent invention with a small molecule that acts as an inhibitoragainst additional tumor-associated proteins including, but not limitedto, EGFR, ErbB2, HER2, and/or VEGF. In certain embodiments, the secondtherapeutic agent is a small molecule that inhibits a cancer stem cellpathway. In some embodiments, the second therapeutic agent is a smallmolecule inhibitor of the Notch pathway. In some embodiments, the secondtherapeutic agent is a small molecule inhibitor of the Wnt pathway. Insome embodiments, the second therapeutic agent is a small moleculeinhibitor of the BMP pathway. In some embodiments, the secondtherapeutic agent is a small molecule that inhibits β-catenin signaling.

In some embodiments, a second therapeutic agent comprises a biologicalmolecule, such as an antibody. For example, treatment can involve thecombined administration of a VEGF/DLL4-binding agent (e.g. an antibody)of the present invention with other antibodies against additionaltumor-associated proteins including, but not limited to, antibodies thatbind EGFR, ErbB2, HER2, and/or VEGF. In certain embodiments, the secondtherapeutic agent is an antibody that is an anti-cancer stem cell markerantibody. In some embodiments, the second therapeutic agent is anantibody that binds a component of the Notch pathway. In someembodiments, the second therapeutic agent is an antibody that binds acomponent of the Wnt pathway. In certain embodiments, the secondtherapeutic agent is an antibody that inhibits a cancer stem cellpathway. In some embodiments, the second therapeutic agent is anantibody inhibitor of the Notch pathway. In some embodiments, the secondtherapeutic agent is an antibody inhibitor of the Wnt pathway. In someembodiments, the second therapeutic agent is an antibody inhibitor ofthe BMP pathway. In some embodiments, the second therapeutic agent is anantibody that inhibits β-catenin signaling. In certain embodiments, thesecond therapeutic agent is an antibody that is an angiogenesisinhibitor or modulator (e.g., an anti-VEGF or VEGF receptor antibody).In certain embodiments, the second therapeutic agent is bevacizumab(AVASTIN), trastuzumab (HERCEPTIN), panitumumab (VECTIBIX), or cetuximab(ERBITUX). Combined administration can include co-administration, eitherin a single pharmaceutical formulation or using separate formulations,or consecutive administration in either order but generally within atime period such that all active agents can exert their biologicalactivities simultaneously.

Furthermore, treatment with a VEGF/DLL4-binding agent described hereincan include combination treatment with other biologic molecules, such asone or more cytokines (e.g., lymphokines, interleukins, tumor necrosisfactors, and/or growth factors) or can be accompanied by surgicalremoval of tumors, cancer cells, or any other therapy deemed necessaryby a treating physician.

In certain embodiments, the treatment involves the administration of aVEGF/DLL4-binding agent (e.g. an antibody) of the present invention incombination with radiation therapy. Treatment with a VEGF/DLL4-bindingagent can occur prior to, concurrently with, or subsequent toadministration of radiation therapy. Dosing schedules for such radiationtherapy can be determined by the skilled medical practitioner.

It will be appreciated that the combination of a VEGF/DLL4-binding agentand an additional therapeutic agent may be administered in any order orconcurrently. Treatment with a VEGF/DLL4-binding agent (e.g., anantibody) can occur prior to, concurrently with, or subsequent toadministration of chemotherapies. Combined administration can includeco-administration, either in a single pharmaceutical formulation orusing separate formulations, or consecutive administration in eitherorder but generally within a time period such that all active agents canexert their biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in The Chemotherapy Source Book, 4^(th) Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

In some embodiments, the VEGF/DLL4-binding agent will be administered topatients that have previously undergone treatment with a secondtherapeutic agent. In certain other embodiments, the VEGF/DLL4-bindingagent and a second therapeutic agent will be administered substantiallysimultaneously or concurrently. For example, a subject may be given aVEGF/DLL4-binding agent (e.g., an antibody) while undergoing a course oftreatment with a second therapeutic agent (e.g., chemotherapy). Incertain embodiments, a VEGF/DLL4-binding agent will be administeredwithin 1 year of the treatment with a second therapeutic agent. Incertain alternative embodiments, a VEGF/DLL4-binding agent will beadministered within 10, 8, 6, 4, or 2 months of any treatment with asecond therapeutic agent. In certain other embodiments, aVEGF/DLL4-binding agent will be administered within 4, 3, 2, or 1 weeksof any treatment with a second therapeutic agent. In some embodiments, aVEGF/DLL4-binding agent will be administered within 5, 4, 3, 2, or 1days of any treatment with a second therapeutic agent. It will furtherbe appreciated that the two (or more) agents or treatments may beadministered to the subject within a matter of hours or minutes (i.e.,substantially simultaneously).

For the treatment of a disease, the appropriate dosage of anVEGF/DLL4-binding agent (e.g., an antibody) of the present inventiondepends on the type of disease to be treated, the severity and course ofthe disease, the responsiveness of the disease, whether theVEGF/DLL4-binding agent or antibody is administered for therapeutic orpreventative purposes, previous therapy, the patient's clinical history,and so on, all at the discretion of the treating physician. TheVEGF/DLL4-binding agent or antibody can be administered one time or as aseries of treatments spread over several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g., reduction in tumor size). Optimal dosing schedules canbe calculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantibody or agent. The administering physician can determine optimumdosages, dosing methodologies, and repetition rates. In certainembodiments, dosage of a VEGF/DLL4-binding agent or antibody is fromabout 0.01 μg to about 100 mg/kg of body weight, from about 0.1 μg toabout 100 mg/kg of body weight, from about 1 μg to about 100 mg/kg ofbody weight, from about 1 mg to about 100 mg/kg of body weight, about 1mg to about 80 mg/kg of body weight from about 10 mg to about 100 mg/kgof body weight, from about 10 mg to about 75 mg/kg of body weight, orfrom about 10 mg to about 50 mg/kg of body weight. In certainembodiments, the dosage of the antibody or other VEGF/DLL4-binding agentis from about 0.1 mg to about 20 mg/kg of body weight. In certainembodiments, dosage can be given once or more daily, weekly, monthly, oryearly. In certain embodiments, the antibody or other VEGF/DLL4-bindingagent is given once every week, once every two weeks, once every threeweeks, or once every month.

In some embodiments, a VEGF/DLL4-binding agent (e.g., an antibody) maybe administered at an initial higher “loading” dose, followed by one ormore lower doses. In some embodiments, the frequency of administrationmay also change. In some embodiments, a dosing regimen may compriseadministering an initial dose, followed by additional doses (or“maintenance” doses) once a week, once every two weeks, once every threeweeks, or once every month. For example, a dosing regimen may compriseadministering an initial loading dose, followed by a weekly maintenancedose of, for example, one-half of the initial dose. Or a dosing regimenmay comprise administering an initial loading dose, followed bymaintenance doses of, for example one-half of the initial dose everyother week. Or a dosing regimen may comprise administering three initialdoses for 3 weeks, followed by maintenance doses of, for example, thesame amount every other week. Or a dosing regimen may compriseadministering an initial dose followed by additional doses every 3 weeksor once a month. The treating physician can estimate repetition ratesfor dosing based on measured residence times and concentrations of thedrug in bodily fluids or tissues. The progress of therapy can bemonitored by conventional techniques and assays.

As is known to those of skill in the art, administration of anytherapeutic agent may lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, drug therapy must be discontinued, and other agentsmay be tried. However, many agents in the same therapeutic class oftendisplay similar side effects and/or toxicities, meaning that the patienteither has to stop therapy, or if possible, suffer from the unpleasantside effects associated with the therapeutic agent.

Side effects from therapeutic agents may include, but are not limitedto, hives, skin rashes, itching, nausea, vomiting, decreased appetite,diarrhea, chills, fever, fatigue, muscle aches and pain, headaches, lowblood pressure, high blood pressure, hypokalemia, low blood counts,bleeding, and cardiac problems.

Thus, one aspect of the present invention is directed to methods oftreating cancer in a patient comprising administering ananti-VEGF/anti-DLL4 bispecific antibody using an intermittent dosingregimen, which may reduce side effects and/or toxicities associated withadministration of the anti-VEGF/anti-DLL4 bispecific antibody. As usedherein, “intermittent dosing” refers to a dosing regimen using a dosinginterval of more than once a week, e.g., dosing once every 2 weeks, onceevery 3 weeks, once every 4 weeks, etc. In some embodiments, a methodfor treating cancer in a human patient comprises administering to thepatient an effective dose of an anti-VEGF/anti-DLL4 bispecific antibodyaccording to an intermittent dosing regimen. In some embodiments, amethod for treating cancer in a human patient comprises administering tothe patient an effective dose of an anti-VEGF/anti-DLL4 bispecificantibody according to an intermittent dosing regimen, and increasing thetherapeutic index of the anti-VEGF/anti-DLL4 bispecific antibody. Insome embodiments, the intermittent dosing regimen comprisesadministering an initial dose of an anti-VEGF/anti-DLL4 bispecificantibody to the patient, and administering subsequent doses of theanti-VEGF/anti-DLL4 bispecific antibody about once every 2 weeks. Insome embodiments, the intermittent dosing regimen comprisesadministering an initial dose of an anti-VEGF/anti-DLL4 bispecificantibody to the patient, and administering subsequent doses of theanti-VEGF/anti-DLL4 bispecific antibody about once every 3 weeks. Insome embodiments, the intermittent dosing regimen comprisesadministering an initial dose of an anti-VEGF/anti-DLL4 bispecificantibody to the patient, and administering subsequent doses of theanti-VEGF/anti-DLL4 bispecific antibody about once every 4 weeks.

In some embodiments, the subsequent doses in an intermittent dosingregimen are about the same amount or less than the initial dose. Inother embodiments, the subsequent doses are a greater amount than theinitial dose. As is known by those of skill in the art, doses used willvary depending on the clinical goals to be achieved. In someembodiments, the initial dose is about 0.25 mg/kg to about 20 mg/kg. Insome embodiments, the initial dose is about 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In certainembodiments, the initial dose is about 0.5 mg/kg. In certainembodiments, the initial dose is about 1 mg/kg. In certain embodiments,the initial dose is about 2.5 mg/kg. In certain embodiments, the initialdose is about 5 mg/kg. In certain embodiments, the initial dose is about7.5 mg/kg. In certain embodiments, the initial dose is about 10 mg/kg.In certain embodiments, the initial dose is about 12.5 mg/kg. In certainembodiments, the initial dose is about 15 mg/kg. In certain embodiments,the initial dose is about 20 mg/kg. In some embodiments, the subsequentdoses are about 0.25 mg/kg to about 15 mg/kg. In certain embodiments,the subsequent doses are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 mg/kg. In certain embodiments, the subsequent doses areabout 0.5 mg/kg. In certain embodiments, the subsequent doses are about1 mg/kg. In certain embodiments, the subsequent doses are about 2.5mg/kg. In certain embodiments, the subsequent doses are about 5 mg/kg.In some embodiments, the subsequent doses are about 7.5 mg/kg. In someembodiments, the subsequent doses are about 10 mg/kg. In someembodiments, the subsequent doses are about 12.5 mg/kg.

In some embodiments, the intermittent dosing regimen comprises: (a)administering to the patient an initial dose of an anti-VEGF/anti-DLL4bispecific antibody of about 2.5 mg/kg and (b) administering subsequentdoses of about 2.5 mg/kg once every 2 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the patientan initial dose of an anti-VEGF/anti-DLL4 bispecific antibody of about 5mg/kg and (b) administering subsequent doses of about 5 mg/kg once every2 weeks. In some embodiments, the intermittent dosing regimen comprises:(a) administering to the patient an initial dose of ananti-VEGF/anti-DLL4 bispecific antibody of about 2.5 mg/kg and (b)administering subsequent doses of about 2.5 mg/kg once every 3 weeks. Insome embodiments, the intermittent dosing regimen comprises: (a)administering to the patient an initial dose of an anti-VEGF/anti-DLL4bispecific antibody of about 5 mg/kg and (b) administering subsequentdoses of about 5 mg/kg once every 3 weeks. In some embodiments, theintermittent dosing regimen comprises: (a) administering to the patientan initial dose of an anti-VEGF/anti-DLL4 bispecific antibody of about2.5 mg/kg and (b) administering subsequent doses of about 2.5 mg/kg onceevery 4 weeks. In some embodiments, the intermittent dosing regimencomprises: (a) administering to the patient an initial dose of ananti-VEGF/anti-DLL4 bispecific antibody of about 5 mg/kg and (b)administering subsequent doses of about 5 mg/kg once every 4 weeks. Incertain embodiments, the initial dose and the maintenance doses aredifferent, for example, the initial dose is about 5 mg/kg and thesubsequent doses are about 2.5 mg/kg. In certain embodiments, anintermittent dosing regimen may comprise a loading dose, for example,the initial dose is about 20 mg/kg and the subsequent doses are about2.5 mg/kg or about 5 mg/kg administered once every 2 weeks, once every 3weeks, or once every 4 weeks.

Another aspect of the present invention is directed to methods forreducing toxicity of an anti-VEGF/anti-DLL4 bispecific antibody in ahuman patient comprises administering to the patient theanti-VEGF/anti-DLL4 bispecific antibody using an intermittent dosingregimen. Another aspect of the present invention is directed to methodsfor reducing side effects of an anti-VEGF/anti-DLL4 bispecific antibodyin a human patient comprises administering to the patient theanti-VEGF/anti-DLL4 bispecific antibody using an intermittent dosingregimen. Another aspect of the present invention is directed to methodsfor increasing the therapeutic index of an anti-VEGF/anti-DLL4bispecific antibody in a human patient comprises administering to thepatient the anti-VEGF/anti-DLL4 bispecific antibody using anintermittent dosing regimen.

The choice of delivery method for the initial and subsequent doses ismade according to the ability of the animal or human patient to tolerateintroduction of the anti-VEGF/anti-DLL4 bispecific antibody into thebody. Thus, in any of the aspects and/or embodiments described herein,the administration of the anti-VEGF/anti-DLL4 bispecific antibody may beby intravenous injection or intravenously. In some embodiments, theadministration is by intravenous infusion. In any of the aspects and/orembodiments described herein, the administration of theanti-VEGF/anti-DLL4 bispecific antibody may be by a non-intravenousroute.

V. Kits Comprising VEGF/DLL4-Binding Agents

The present invention provides kits that comprise the VEGF/DLL4-bindingagents (e.g., antibodies) described herein and that can be used toperform the methods described herein. In certain embodiments, a kitcomprises at least one purified antibody against VEGF and/or DLL4 in oneor more containers. In some embodiments, the kits contain all of thecomponents necessary and/or sufficient to perform a detection assay,including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results. One skilledin the art will readily recognize that the disclosed VEGF/DLL4-bindingagents of the present invention can be readily incorporated into one ofthe established kit formats which are well known in the art.

Further provided are kits comprising a VEGF/DLL4-binding agent (e.g., ananti-VEGF/anti-DLL4 bispecific antibody), as well as at least oneadditional therapeutic agent. In certain embodiments, the second (ormore) therapeutic agent is a chemotherapeutic agent. In certainembodiments, the second (or more) therapeutic agent is an angiogenesisinhibitor.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the present disclosure.

EXAMPLES Example 1 Binding Affinities of Anti-VEGF/Anti-DLL4 Antibodies

The K_(D)s of parental antibodies anti-VEGF 219R45 (IgG format),anti-DLL4 21R79 (IgG format), anti-DLL4 21M18 (IgG format) andbispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79 weredetermined using a Biacore 2000 system from Biacore LifeSciences (GEHealthcare). Recombinant human DLL4-Fc or mouse DLL4-Fc proteins wereimmobilized on CM5 carboxyl chips using standard amine-based chemistry(NHS/EDC) and blocked with ethanolamine. Recombinant human VEGF₁₆₅ ormouse VEGF₁₆₅ were biotinylated and immobilized on streptavidin chips.The antibodies were serially diluted 2-fold from 100 nM to 0.78 nM inHBS-P (0.01M HEPES pH7.4, 0.15M NaCl, 0.005% v/v Polysorbate 20). Foreach antibody, all 8 dilutions were sequentially injected over aspecific chip. Kinetic data were collected over time and were fit usingthe simultaneous global fit equation to yield affinity constants (K_(D)values) for each bispecific antibody.

TABLE 3 hVEGF mVEGF hDLL4 mDLL4 Antibody (nM) (nM) (nM) (nM) 219R45 0.6722.9 NB NB 21M18 NB NB <0.1 NB 21R79 NB NB <0.1 NB 219R45-MB-21M18 0.3625.5 16 NB 219R45-MB-21R79 0.68 12.5 0.53 NB

As shown in Table 3, bispecific antibody 219R45-MB-21M18 had an affinityconstant (K_(D)) for human VEGF of 0.36 nM and a K_(D) for human DLL4 of16 nM. Bispecific antibody 219R45-MB-21R79 had a K_(D) for human VEGF of0.68 nM and a K_(D) for human DLL4 of 0.53 nM. Both bispecificantibodies demonstrated weaker binding to mouse VEGF as compared tohuman VEGF and neither antibody bound mouse DLL4. Thus, both bispecificantibodies demonstrated similar binding affinity to human VEGF and219R45-MB-21R79 demonstrated approximately 30-fold stronger binding tohuman DLL4 than 219R45-MB-21M18. Furthermore, bispecific antibody219R45-MB-21R79 had a similar binding affinity to human VEGF despite thefact the bispecific antibody is monovalent for VEGF as compared to thebivalent parental antibody.

Several additional anti-DLL4 antibodies were identified that had bindingaffinities intermediate to the K_(D)s of 21M18 and 21R79. Two of theseanti-DLL4 antibodies were used to produce anti-VEGF/anti-DLL4 bispecificantibodies 219R45-MB-21R75 and 219R45-MB-21R83. Using the Biacore 2000system as described above, the K_(D)s of the bispecific antibodies219R45-MB-21R75 and 219R45-MB-21R83 to human DLL4 were determined. Acomparison of the binding affinity to human DLL4 of these fouranti-VEGF/anti-DLL4 bispecific antibodies is shown in Table 4.

The CDRs for anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18,219R45-MB-21R79, 219R45-MB-21R75, and 219R45-MB-21R83 are shown in FIG.1A. The heavy chain and light chain variable region SEQ ID NOs are shownin FIG. 1B and the heavy chain and light chain SEQ ID NOs (with andwithout signal sequence) are shown in FIG. 1C.

Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21M18 comprises a (a)heavy chain encoded by the DNA comprising SEQ ID NO:75 deposited withAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., USA, under the conditions of the Budapest Treaty on Sep.21, 2012 and assigned designation number PTA-13233, (b) a heavy chainencoded by the DNA comprising SEQ ID NO:33 deposited with ATCC under theconditions of the Budapest Treaty on Sep. 21, 2012 and assigneddesignation number PTA-13236, and (c) a light chain encoded by the DNAcomprising SEQ ID NO:34 deposited with ATCC under the conditions of theBudapest Treaty on Sep. 21, 2012 and assigned designation numberPTA-13235.

Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R79 comprises a (a)heavy chain encoded by the DNA comprising SEQ ID NO:31 deposited withATCC under the conditions of the Budapest Treaty on Sep. 21, 2012 andassigned designation number PTA-13232, (b) a heavy chain encoded by theDNA comprising SEQ ID NO:33 deposited with ATCC under the conditions ofthe Budapest Treaty on Sep. 21, 2012 and assigned designation numberPTA-13236, and (c) a light chain encoded by the DNA comprising SEQ IDNO:34 deposited with ATCC under the conditions of the Budapest Treaty onSep. 21, 2012 and assigned designation number PTA-13235.

Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R83 comprises (a) aheavy chain encoded by the DNA comprising SEQ ID NO:72 deposited withATCC under the conditions of the Budapest Treaty on Oct. 24, 2012 andassigned designation number PTA-13278, (b) a heavy chain encoded by theDNA comprising SEQ ID NO:33 deposited with ATCC under the conditions ofthe Budapest Treaty on Sep. 21, 2012 and assigned designation numberPTA-13236, and (c) a light chain encoded by the DNA comprising SEQ IDNO:34 deposited with ATCC under the conditions of the Budapest Treaty onSep. 21, 2012 and assigned designation number PTA-13235.

Anti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21R75 comprises (a) aheavy chain encoded by the DNA comprising SEQ ID NO:74 deposited withATCC under the conditions of the Budapest Treaty on Sep. 21, 2012 andassigned designation number PTA-13234, (b) a heavy chain encoded by theDNA comprising SEQ ID NO:33 deposited with ATCC under the conditions ofthe Budapest Treaty on Sep. 21, 2012 and assigned designation numberPTA-13236, and (c) a light chain encoded by the DNA comprising SEQ IDNO:34 deposited with ATCC under the conditions of the Budapest Treaty onSep. 21, 2012 and assigned designation number PTA-13235.

TABLE 4 Antibody Heavy chain CDR2 hDLL4 (nM) 219R45-MB-21M18YISSYNGATNYNQKFKG 16.00 SEQ ID NO: 15) 219R45-MB-21R79 YIANYNRATNYNQKFKG0.53 (SEQ ID NO: 14) 219R45-MB-21R75 YIAGYKDATNYNQKFKG 5.10(SEQ ID NO: 59) 219R45-MB-21R83 YISNYNRATNYNQKFKG 1.30 (SEQ ID NO: 65)

Example 2 HTRF Assay for Simultaneous Binding of Bispecific Antibodiesto Human VEGF and Human DLL4

To characterize the binding capabilities of certain antibodies and/orantibody mixtures to both VEGF and DLL4, homogeneous time resolvedfluorescence (HTRF) assays were performed. Antibodies tested wereanti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18 and219R45-MB-21R79, parental antibodies 219R45 (anti-VEGF), 21M18(anti-DLL4), 21R79 (anti-DLL4), a combination of 219R45 and 21M18, or acombination of 219R45 and 21R79. The antibodies or antibody mixtureswere serially diluted 2-fold from 3000 nM to 2.9 nM in binding buffer(1×PBS, 0.1% gelatin, 0.1% Polysorbate 20, 400 mM potassium fluoride)and placed in a white 96-well plate. An equal volume of solutioncontaining 4 μg/ml of d2-labeled hDLL4-Fc and 21.4 ng/ml Europiumcrypate-labeled hVEGF₁₆₅ was added to each well for a final volume of100 μl (final concentrations of acceptor and donor fluorophores were 2μg/ml and 10.7 ng/ml, respectively). The assay plates were incubated for2 hours to overnight and read on a SpectraMax M5e Microplate reader(Molecular Devices, Sunnyvale Calif.) at an excitation wavelength of 314nm.

As shown in FIG. 2 , anti-VEGF/anti-DLL4 bispecific antibodies219R45-MB-21M18 and 219R45-MB-21R79, were able to bind both hVEGF andhDLL4 simultaneously. Importantly, neither of the combinations of theparental antibodies (i.e., 219R45 and 21M18 or 219R45 and 21R79) wasable to bind VEGF and DLL4 simultaneously. These results clearlydemonstrate that the anti-VEGF/anti-DLL4 bispecific antibodies219R45-MB-21M18 and 219R45-MB-21R79 are capable of functioningdifferently than just a mixture of the two individual antibodies.

Example 3 Inhibition of HUVEC Proliferation by Anti-VEGF/Anti-DLL4Bispecific Antibodies

HUVEC cells were obtained from Lonza (Walkersville Md.) and cultured ingrowth media (M199, 10% heat-inactivated FBS (HI-FBS), 50 μg/ml EGS, 1×heparin, 1 mM L-glutamine). For the HUVEC proliferation assay, a 96-wellplate was pre-coated with 50 μl of 10 μg/ml rat tail collagen type Isolution (collagen I in 0.02N acetic acid) and incubated at 4° C.overnight. After incubation, the plate was thoroughly aspirated toremove unbound collagen I solution and washed once with 200 μl DPBS. TheHUVEC cells were removed from the surface of the growth flasks using anendothelial cell subclone reagent and centrifuged at 1200 rpm for 5minutes at 4° C. The cells were resuspended in starvation/assay medium(M199 and 2% HI-FBS, 1× heparin, 5 U/ml heparin-glutamine) at a densityof 10⁵ cells/ml. The cells were seeded into the collagen-coated assayplate at 5000 cells/well, 50 ul/well. The cells were incubated for 3hours at 37° C., washed one time, refed with 100 ul assay media, andincubated overnight at 37° C. The next day, bispecific antibodies219R45-MB-21M18, 219R45-MB-21R79, parental antibody 219R45, or controlantibody LZ1 were prepared in a mixture with human VEGF (R&D Biosystems,Minneapolis Minn.). The antibodies were serially diluted 5-fold from 20μM to 0.25 nM in assay buffer in combination with hVEGF (finalconcentration 5 ng/ml). The mixture was pre-incubated at 37° C. for 2hours. The medium was removed from the assay plate, and 100 μl of theantibody/hVEGF mixture was added to each well. After 3-4 daysincubation, medium was removed and a fresh aliquot of the antibody/hVEGFmixture was added to each well and allowed to incubate for another 4days. On day 7, 20 μl of Alamar Blue reagent (Invitrogen, Carlsbad,Calif.) was added to each well and incubated at 37° C. for 5-6 hours.The plate was read with a SpectraMax M5e Microplate reader (MolecularDevices, Sunnyvale Calif.) using a excitation wavelength of 539 nm andan emission wavelength of 590 nm.

As shown in FIG. 3 , anti-VEGF/anti-DLL4 bispecific antibodies219R45-MB-21M18 and 219R45-MB-21R79, as well as parental anti-VEGFantibody 219R45 inhibited HUVEC proliferation. These resultsdemonstrated that the bispecific antibodies were capable of inhibitingVEGF-induced proliferation of HUVEC cells.

Example 4 Inhibition of DLL4-Induced Notch Signalling by BispecificAntibodies

Human PC3 cells were transfected with an expression vector encoding afull-length human Notch2 receptor and a firefly luciferase reportervector (8xCBF-luciferase reporter) that is responsive to Notchsignaling. The cells were also transfected with a Renilla luciferasereporter (Promega, Madison Wis.) as an internal control for transfectionefficiency. Purified human DLL4 protein was coated onto 96-well platesat 100 ng/well and Notch2-expressing PC3-luc cells were added to thewells. Anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18,219R45-MB-21R79, parental anti-DLL4 antibodies 21M18, 21R79 or a controlantibody LZ1 were serially diluted 5-fold from 20 ug/ml to 0.064 ug/ml,added to the appropriate wells, and incubated overnight. Luciferaseactivity was determined using a dual luciferase assay kit (Promega,Madison, Wis.) with firefly luciferase activity normalized to Renillaluciferase activity.

As shown in FIG. 4 , anti-VEGF/anti-DLL4 bispecific antibody219R45-MB-21R79 and parental anti-DLL4 antibodies 21M18 and 21R79inhibited DLL4-induced Notch signaling. Bispecific antibody219R45-MB-21M18 inhibited DLL4-induced Notch signaling only at highantibody concentrations. These results demonstrated that bispecificantibody 219R45-MB-21R79, and to a lesser extent bispecific antibody219R45-MB-21M18, were capable of inhibiting DLL4-induced Notchsignaling. Thus, in combination with the results presented in Example 3,the anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21R79 and219R45-MB-21M18 have demonstrated the ability to inhibit bothVEGF-induced and DLL4-induced signaling and/or proliferation functions.

Example 5 Inhibition of Tumor Growth In Vivo by a Bispecific Antibody ina Human Skin Graft Model

A human skin graft model has been reported which comprises a human skingraft and human tumor cells. A human skin graft is established and thenhuman tumor cells are implanted into the skin graft, allowing the tumorcells to grow in an environment with human stroma and vasculature(Tahtis et al., 2003, Mol. Cancer Ther. 2:229-737). Human skin sampleswere obtained from neonatal foreskin tissue and grafted onto the lateralflank of NOD-SCID mice. After establishment of the skin graft,luciferase-labeled OMP-C8 colon tumor cells (20,000 cells) were injectedintradermally into the human skin. Tumor growth was monitored bybioluminescence imaging using an IVIS imaging system (Caliper LifeSciences, Mountain View, Calif.). Tumors were allowed to grow until theyreached 1.2×10⁶ photons per second. Tumor-bearing mice (n=6 mice/group)were randomized and treated with control Ab, anti-hDLL4 antibody 21M18,anti-VEGF antibody bevacizumab, or anti-VEGF/anti-DLL4 bispecificantibody 219R45-MB-21M18. Animals were treated once a week andantibodies were administered intraperitoneally at a dose of 25 mg/kg.Tumor growth was monitored by bioluminescence imaging on the indicateddays.

As shown in FIG. 5 , both anti-hDLL4 antibody 21M18 and anti-VEGFantibody bevacizumab inhibited tumor growth in this human skingraft/human tumor model. Furthermore, bispecific anti-VEGF/anti-DLL4bispecific antibody 219R45-MB-21M18 was more effective than either theanti-DLL4 antibody or the anti-VEGF antibody alone. These datademonstrate the utility of simultaneously targeting DLL4 and VEGF with abispecific antibody.

Example 6

Tumorigenicity of OMP-PN8 Pancreatic Tumor Cells after Treatment withAnti-VEGF/Anti-DLL4 Bispecific Antibodies

Mice bearing OMP-PN8 pancreatic tumors were treated with controlantibody (15 mg/kg), anti-hDLL4 antibody 21M18 (15 mg/kg), anti-VEGFantibody bevacizumab (15 mg/kg), or anti-VEGF/anti-DLL4 bispecificantibodies 219R45-MB-21M18 or 219R45-MB-21R79 (30 mg/kg) with or withoutgemcitabine (70 mg/kg). Following four weeks of treatment, tumors wereharvested, processed to single cell suspensions and the human tumorcells were purified by immunomagnetic depletion of murine cells. 90human tumor cells from each treatment group were transferred to a newcohort of mice (n=10 mice/group). Tumors were allowed to grow for 55days without any treatment and tumor volumes were measured withelectronic calipers.

FIG. 6 shows the tumor volume from the individual mice in each group.Cells isolated from mice treated with anti-hDLL4 antibody 21M18 hadgreatly decreased tumorigenicity, 5 out of 10 mice had tumors, ascompared to cells isolated from mice treated with control antibody where9 out of 10 mice had tumors. The reduction in tumor growth frequencyindicates a reduction in cancer stem cell frequency. In contrast,bevacizumab treatment resulted in no reduction of tumor growthfrequency, 10 out of 10 mice had tumors. Similar to bevacizumab,treatment with gemcitabine as a single agent had no effect on tumorgrowth frequency as 10 out of 10 mice had tumors. Theanti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18 and219R45-MB-21R79 both reduced tumor growth frequency (5 out of 10 micehad tumors and 4 out of 10 mice had tumors, respectively). Combinationtreatment with gemcitabine appeared to have no effect on tumor growthfrequency. These data indicate that targeting DLL4 reduces cancer stemcell frequency while targeting VEGF alone does not. Importantly, thesedata indicate that the anti-CSC activity of the anti-DLL4 antibody isretained in a bispecific antibody.

Example 7 Bispecific Antibody ELISA

VEGF (ATGEN, South Korea) was coated onto Nunc maxisorb plates at 2ug/ml (100 μl/well) and incubated overnight at 2-8° C. Bispecificantibodies 219R45-MB-21M18, 219R45-MB-21R79, 219R45-MB-21R75, and219R45-MB-21R83 were diluted in blocking buffer (lx PBS, 0.1% gelatin,0.1% Polysorbate-20, pH 7.4) containing 2 μg/ml biotin-DLL4-hFc. Theantibodies were serially diluted 3-fold from 500 ng/ml to 0.008 ng/ml.The antibody samples were incubated for 2 hours in blocking buffercontaining the biotin-DLL4-hFc. After incubation, the antibody sampleswere transferred to the VEGF-coated assay plate (100 ul/well) andincubated for 2 hours. Streptavidin-HRP (Jackson ImmunoResearch, WestGrove, Pa.) was added to each well and incubated for 1 hr. TMB substratewas added to the wells with a 10 minute color development and thereaction was stopped with 2M sulfuric acid. Absorbance was read at450-650 nm and the data analyzed using the 4-parameter fit within theSoftmax Pro analysis program (Molecular Devices, Sunnyvale, Calif.).

FIG. 7 shows the titration curves of bispecific antibodies219R45-MB-21M18 (open circles), 219R45-MB-21R79 (open squares),219R45-MB-21R75 (open triangles), and 219R45-MB-21R83 (open diamonds) incomparison to a reference anti-VEGF/anti-DLL4 bispecific antibody (solidcircles). Relative potencies for the bispecific antibodies as comparedto the reference bispecific antibody are shown in Table 5.

TABLE 5 Relative Antibody Potency (%) 219R45-MB-21M18 67 219R45-MB-21R79501 219R45-MB-21R75 422 219R45-MB-21R83 222

Bispecific antibody 219R45-MB-21R79 was the most potent, about 7-foldmore potent than 219R45-MB-21M18, which reflected the higher affinity ofthe 21R79 antigen-binding site.

Example 8 Bispecific Antibody Production

Bispecific antibodies were produced using a GS-CHO cell line. CHOK1SVcells (Lonza Biologics) were transfected via electroporation with thegene(s) of interest coupled with glutamine synthetase (GS) as theselectable marker. Transfectants and subclones were screened forantibody productivity and the high producers were selected for scaled-upproduction. Cells were grown using a fed-batch process and fed-batchbioreactors. Accumulated antibody in harvested cell culture fluid (HCCF)was isolated and purified using chromatography techniques.

Bispecific antibody cell lines 219R45-MB-21M18.010.017 and219R45-MB-21R79.017.003 were cultured in 5 L stirred tank bioreactorsfor 14 days. Cell line 219R45-MB-21M18.010.017 produced a final antibodytiter of 3.0 g/L and cell line 219R45-MB-21R79.017.003 produced a finalantibody titer of 0.8 g/L. Cell lines 219R45-MB-21R75.101 and219R45-MB-21R83.113 were cultured in 25 L WAVE bioreactor systems (GEHealthcare) using a fed-batch process that achieved final antibodytiters of 0.4 g/L. Bispecific antibody cell lines219R45-MB-21M18AG.138.007, 219R45-MB-21M18AG.038.009,219R45-MB-21M18AG.142.002, 219R45-MB-21R79AG.072.014 and219R45-MB-21R83AG.129.003 were cultured in 5 L stirred tank bioreactorsfor 14-15 days. Cell line 219R45-MB-21M18AG.138.007 produced a finalantibody titer of 1.0 g/L after 14 days. Cell line219R45-MB-21M18AG.038.009 produced a final antibody titer of 1.6 g/Lafter 14 days. Cell line 219R45-MB-21M18AG.142.002 produced a finalantibody titer of 2.6 g/L after 14 days. Cell line219R45-MB-21R79AG.072.014 produced a final antibody titer of 2.1 g/Lafter 15 days. Cell line 219R45-MB-21M18AG.038.009 produced a finalantibody titer of 2.4 g/L after 15 days. Culture fluid was harvested byfiltration from each of these four cell lines and subjected to Protein Aaffinity chromatography. The Protein A column was washed with a seriesof buffers and the antibodies were eluted using a low pH elution buffer.Initial characterization of the purity of the bispecific antibodies wasperformed using size exclusion chromatography (SEC-HPLC) and isoelectricfocusing (IEF).

Size exclusion chromatography (SEC) was used to determine the purity ofthe antibody product. SEC is a well known chromatographic method inwhich molecules (e.g., antibodies) in solution are separated by theirsize. SEC may be used to distinguish an antibody product from aggregateand/or impurities, and to determine the percentage of the antibodyproduct as compared to the total mixture. As used herein, SEC does notdistinguish between a homomeric antibody and a heterodimeric bispecificantibody.

Imaged capillary isoelectric focusing (icIEF) was used to determineidentity and purity of the bispecific antibody heterodimers. UsingicIEF, the charge isoforms of an antibody are separated according totheir pI and the result is a “fingerprint” of the antibody's chargedistribution. The icIEF method can also serve as a determination ofpurity by separating the bispecific antibody heterodimers by theirdistinct pI from any homodimer products or impurities.

Bispecific antibody samples were analyzed by icIEF on a ProteinSimpleICE280 instrument (ProteinSimple, Santa Clara, Calif.). For thisanalysis, a protein mixture is introduced into a capillary, high voltageis applied across the capillary and ampholytes establish a linear pHgradient along the length of the capillary. Under the influence of theelectric field, the pI markers and the protein mixture both migrate thelength of the capillary until a pH value is reached where the net chargeis zero. Once focused, the ICE280 instrument uses whole-column imagingdetection with a 280-nm UV camera to monitor the pattern of proteinisoforms within the capillary. The resulting electropherogram iscalibrated using internal pI markers and integrated to establish therespective percentage areas of the different charged isoforms of theprotein mixture. The charge profiles from several anti-VEGF/anti-DLL4bispecific antibodies are shown in FIG. 8 . For this experiment, ProteinA eluates were diluted with MilliQ water to a concentration of 6.6mg/ml. A total of 18 μl of the sample was mixed with 100 μL of 8M urea,70 μl of 0.5% methylcellulose, 8 μL of 3-10 Pharmalyte, 2 μL of high pImarker and 2 μL of low pI marker to a final volume of 200 μl. Table 6shows the percentage of antibody product from cell lines219R45-MB-21M18.010.017, 219R45-MB-21R79.017.002, 219R45-MB-21R75.101,219R45-MB-21R83.113, 219R45-MB-21M18.138.007, 219R45-MB-21M18AG.038.009,219R45-MB-21M18AG.142.002, 219R45-MB-21R79AG.072.014, and219R45-MB-21R83AG.129.003 after Protein A affinity chromatography asdetermined by SEC-HPLC. Table 6 also shows the percentage ofheterodimeric antibodies from cell lines 219R45-MB-21M18.010.017,219R45-MB-21R79.017.002, 219R45-MB-21R75.101, 219R45-MB-21R83.113,219R45-MB-21M18.138.007, 219R45-MB-21M18AG.038.009,219R45-MB-21M18AG.142.002, 219R45-MB-21R79AG.072.014, and219R45-MB-21R83AG.129.003 after Protein A affinity chromatography asanalyzed by icIEF.

TABLE 6 Purity by Antibody Purity by IEF (% Cell Line Titer (g/L) SEC(%) heterodimer) 219R45-MB-21M18.010.017 3.0 73.9 47.2219R45-MB-21R79.017.002 0.8 79.3 72.5 219R45-MB-21R75.101 0.4 91.2 84.9219R45-MB-21R83.113 0.4 91.8 91.4 219R45-MB-21M18.138.007 1.0 92.6 95.8219R45-MB-21M18AG.038.009 1.6 89.6 89.0 219R45-MB-21M18AG.142.002 2.691.2 84.6 219R45-MB-21R79AG.072.014 2.1 87.8 84.9219R45-MB-21R83AG.129.003 2.4 89.4 90.5

The purity of the bispecific antibody product can be increased furtherby additional chromatography steps. After Protein A affinitychromatography, the eluate fraction was held at a low pH for no lessthan 60 minutes at room temperature for viral inactivation. The antibodysolution (Protein A column eluate, pH adjusted) was loaded onto a stronganion-exchange column. Product- and process-related impurities bound tothe anion exchange chromatography resin and the flow-through fraction(antibody product) was collected. In some cases, purity was furtherimproved by use of a multi-modal chromatography resin such as ceramichydroxyapatite. In some cases, buffer exchange of the antibody productwas undertaken using ultrafiltration and diafiltration techniques, afterwhich excipients were added. The formulated antibody was sterilefiltered into sterile containers and stored refrigerated or frozen.Purity of the bispecific antibodies was re-assessed using SEC-HPLC andIEF.

TABLE 7 Purity by Purity by IEF Cell Line SEC (%) (% heterodimer)219R45-MB-21M18.010.017 98.9 98.5 219R45-MB-21R79.017.002 95.1 99.3219R45-MB-21R75.101 97.2 98.2 219R45-MB-21R83.113 95.3 91.4219R45-MB-21M18.138.007 98.1 100 219R45-MB-21M18AG.142.002 99.6 100219R45-MB-21R79AG.072.014 98.2 100 219R45-MB-21R83AG.129.003 998.6 100

As shown in Table 7, the purification of the anti-VEGF/anti-DLL4bispecific antibodies with additional chromatography steps after ProteinA resulted in isolation of antibody products that were 95% to about 99%pure as analyzed by SEC. Analysis by IEF determined that purifiedanti-VEGF/anti-DLL4 bispecific antibody from cell line219R45-MB-21M18.010.017 was 98.5% heterodimeric, anti-VEGF/anti-DLL4bispecific antibody from cell line 219R45-MB-21R79.017.002 was 99.3%heterodimeric, anti-VEGF/anti-DLL4 bispecific antibody from cell line219R45-MB-21R75.101 was 98.2% heterodimeric, anti-VEGF/anti-DLL4bispecific antibody from cell line 219R45-MB-21R83.113 was 91.4%heterodimeric, anti-VEGF/anti-DLL4 bispecific antibody from cell line219R45-MB-21M18.138.007 was 100% heterodimeric, anti-VEGF/anti-DLL4bispecific antibody from cell line 219R45-MB-21M18AG.142.002 was 100%heterodimeric, anti-VEGF/anti-DLL4 bispecific antibody from cell line219R45-MB-21R79AG.072.014 was 100% heterodimeric, andanti-VEGF/anti-DLL4 bispecific antibody from cell line219R45-MB-21R83AG.129.003 was 100% heterodimeric. These resultsdemonstrated that the anion-exchange chromatography step greatlyincreased the percentage of heterodimeric antibodies as compared topurification with Protein A chromatography alone. The addition of amulti-modal chromatography step such as ceramic hydroxyapatite can alsoimprove monomeric purity (as determined by SEP-HPLC).

Example 9 Inhibition of OMP-C8 Colon Tumor Growth In Vivo TumorRecurrence Model

Single cell suspensions of OMP-C8 colon tumor xenografts (20,000 cells)were injected subcutaneously into the flanks of 6-8 week old NOD/SCIDmice. Tumors were allowed to grow for 33 days until they reached anaverage volume of 240 mm³. The mice were randomized (n=10 per group) andtreated with anti-hDLL4 antibody 21M18, anti-VEGF antibody bevacizumab,a combination of antibodies 21M18 and bevacizumab, anti-VEGF/anti-DLL4bispecific antibody 219R45-MB-21M18, anti-VEGF/anti-DLL4 bispecificantibody 219R45-MB-21R79, or control antibody, all in combination withirinotecan. Antibodies and irinotecan were dosed weekly by injectioninto the intraperitoneal cavity. Antibodies 21M18 and bevacizumab weredosed at 7.5 mg/kg, bispecific antibodies 219R45-MB-21M18 and219R45-MB-21R79 were dosed at 15 mg/kg, and irinotecan was dosed at 45mg/kg. Irinotecan was dosed for four weeks, at which time, it wasdiscontinued and the administration of the antibodies continued. Tumorgrowth was monitored and tumor volumes were measured with electroniccalipers at the indicated time points. Data are expressed as mean±S.E.M.

As shown in FIG. 9 , anti-hDLL4 antibody 21M18 continued to inhibittumor growth after treatment with irinotecan was stopped. In contrast,anti-VEGF antibody bevacizumab was not able to inhibit regrowth of thetumor after irinotecan had been stopped. The combination of anti-DLL4antibody 21M18 and anti-VEGF antibody bevacizumab resulted in greaterinhibition of tumor regrowth than either agent alone. Furthermore, theanti-VEGF/anti-DLL4 bispecific antibody 219R45-MB-21M18 was moreeffective at inhibiting tumor regrowth than the mixture of the twoantibodies.

Example 10 Reduction in Tumorigenicity of OMP-C8 Colon Tumors

Single cell suspensions of OMP-C8 colon tumor xenografts (20,000 cells)were injected subcutaneously into the flanks of 6-8 week old NOD/SCIDmice. Tumors were allowed to grow for 33 days until they reached anaverage volume of 300 mm³. The mice were randomized (n=5 per group) andtreated with anti-DLL4 antibody 21M18, anti-VEGF antibody bevacizumab, acombination of antibodies 21M18 and bevacizumab, anti-VEGF/anti-DLL4bispecific antibody 219R45-MB-21M18, anti-VEGF/anti-DLL4 bispecificantibody 219R45-MB-21R79, or control antibody, either in combinationwith irinotecan or without irinotecan. Antibodies and irinotecan weredosed weekly by injection into the intraperitoneal cavity. Antibodies21M18 and bevacizumab were dosed at 7.5 mg/kg, bispecific antibodies219R45-MB-21M18 and 219R45-MB-21R79 were dosed at 15 mg/kg, andirinotecan was dosed at 45 mg/kg. Tumors were harvested after 4 weeks,processed into single cell suspensions, and the human tumor cells wereisolated. 150 tumor cells from each experimental group were injectedsubcutaneously into a new cohort of mice (n=10 per group) and tumorswere allowed to grow without treatment. Tumor growth was monitored andtumor volumes were measured with electronic calipers.

Individual tumor volumes at day 68 are shown in FIG. 10 . Anti-DLL4antibody 21M18, the combination of 21M18 with anti-VEGF antibodybevacizumab, bispecific antibodies 219R45-MB-21M18 and 219R45-MB-21R79,and irinotecan all reduced tumor growth frequency as single agents. Incontrast, anti-VEGF bevacizumab as a single agent had no effect on tumorgrowth frequency as compared to the control antibody. In the groupstreated with a combination of irinotecan and antibodies, the bispecificantibody 219R45-MB-21M18 had the greatest effect in reducing tumorgrowth frequency.

Example 11 Inhibition of OMP-C8 Colon Tumor Growth In Vivo

Single cell suspensions of OMP-C8 colon tumor xenografts (50,000 cells)were injected subcutaneously into the flanks of 6-8 week old NOD/SCIDmice. Tumors were allowed to grow for 21 days until they reached anaverage volume of 80 mm³. The mice were randomized (n=8 per group) andtreated with anti-DLL4 antibody 21M18, anti-VEGF antibody bevacizumab,anti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18,219R45-MB-21R75, 219R45-MB-21R79, 219R45-MB-21R83, or control antibody,either alone or in combination with irinotecan. Antibodies andirinotecan were dosed weekly by injection into the intraperitonealcavity. Bevacizumab and bispecific antibodies 219R45-MB-21M18,219R45-MB-21R75, 219R45-MB-21R79, and 219R45-MB-21R83 were dosed at 15mg/kg, and irinotecan was dosed at 7.5 mg/kg. Tumor growth was monitoredand tumor volumes were measured with electronic calipers at theindicated time points. Data are expressed as mean±S.E.M.

As single agents, all four anti-VEGF/anti-DLL4 bispecific antibodiesshowed enhanced anti-tumor activity relative to anti-VEGF antibodybevacizumab (FIG. 11A). In combination with irinotecan, treatment withanti-VEGF/anti-DLL4 bispecific antibodies 219R45-MB-21M18 and219R45-MB-21R83 resulted in the greatest inhibition of tumor growth(FIG. 11B).

Following the treatment phase, tumor sections were prepared and analyzedby hematoxylin and eosin (H&E) staining. The tumors treated with219R45-MB-21M18 and 219R45-MB-21R83 in combination with irinotecanshowed dark pink staining regions providing evidence of extensivecalcification. This is characteristic of highly necrotic tumor tissue.

Example 12 Non-GLP Toxicity Study of Bispecific Antibodies in CynomolgusMonkeys

A non-GLP toxicity study in cynomolgus monkeys was initiated to evaluateand compare the toxicity profile of some of the bispecific antibodies.The animals were dosed with 0 mg/kg (control), 5 mg/kg (low dose), or 30mg/kg (high dose) of anti-DLL4/anti-VEGF bispecific antibody(219R45-MB-21M18, 219R45-MB-21R83, or 219R45-MB-21R79) every 2 weeks viaIV infusion. 3 males and 3 females were dosed in each group. After 15weeks, mean body weights were lower in animals receiving the high doseof 219R45-MB-21R79 than in animals that received the high dose of either219R45-MB-21R18 or 219R45-MB-21R83. In addition, mean serum albuminlevels were lower in animals that received 219R45-MB-21R79 than in thosethat received either 219R45-MB-21R18 or 219R45-MB-21R83. Althoughpreliminary in nature, these early data suggest that 219R45-MB-21R18 and219R45-MB-21R83 may have a superior toxicity profile compared to219R45-MB-21R79.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences including both polynucleotide andpolypeptide sequences cited herein are hereby incorporated by referenceherein in their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence was specifically and individuallyindicated to be so incorporated by reference.

SEQUENCES21M18 Heavy chain with signal sequence (underlined) (SEQ ID NO: 1)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R79 Heavy chain with signal sequence (underlined) (SEQ ID NO: 2)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK219R45 Heavy chain with signal sequence (underlined) (SEQ ID NO: 3)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLight chain with signal sequence (underlined) (SEQ ID NO: 4)MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC21M18 Heavy chain without predicted signal sequence (SEQ ID NO: 5)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R79 Heavy chain without predicted signal sequence (SEQ ID NO: 6)NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK219R45 Heavy chain without predicted signal sequence (SEQ ID NO: 7)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKLight chain without predicted signal sequence (SEQ ID NO: 8)DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKRTVAAPSVIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC21M18 Heavy chain variable region (SEQ ID NO: 9)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21R79 Heavy chain variable region (SEQ ID NO: 10)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS219R45 Heavy chain variable region (SEQ ID NO: 11)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSSLight chain variable region (SEQ ID NO: 12)DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK21R75, 21R79, 21R83, and 21M18 Heavy chain CDR1 (SEQ ID NO: 13) TAYYIHAlternative 21R75, 21R79, 21R83, and 21M18 Heavy chain CDR1 (SEQ ID NO: 79)AYYIH 21R79 Heavy chain CDR2 (SEQ ID NO: 14) YIANYNRATNYNQKFKG21M18 Heavy chain CDR2 (SEQ ID NO: 15) YISSYNGATNYNQKFKG21R75, 21R79, 21R83, and 21M18 Heavy chain CDR3 (SEQ ID NO: 16)RDYDYDVGMDY 219R45 Heavy chain CDR1 (SEQ ID NO: 17) NYWMH219R45 Heavy chain CDR2 (SEQ ID NO: 18) DINPSNGRTSYKEKFKR219R45 Heavy chain CDR3 (SEQ ID NO: 19) HYDDKYYPLMDYLight chain CDR1 (SEQ ID NO: 20) RASESVDNYGISFMKLight chain CDR2 (SEQ ID NO: 21) AASNQGSLight chain CDR3 (SEQ ID NO: 22) QQSKEVPWTFGGHuman DLL4 with signal sequence (underlined) (SEQ ID NO: 23)MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGHuman DLL4 without predicted signal sequence (SEQ ID NO: 24)SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTSIDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVG Human DLL4 N-Terminal Region (SEQ ID NO: 25)SGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVCLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLITEAWHAPGDDLRPEALPPDALISKIAIQ GSLAVGQNHuman DLL4 DSL Domain (SEQ ID NO: 26)WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTG EYCHuman VEGF-A with signal sequence (underlined)(SEQ ID NO: 27)MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRHuman VEGF-A without predicted signal sequence (SEQ ID NO: 28)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR21M18 Heavy chain nucleotide sequence (13B Version 1) (SEQ ID NO: 29)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGGTCAAGCAGGCCCCTGGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGCGCCACCAACTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCTGCCTCCACCAAGGGCCCATCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCTGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTAG21R79 Heavy chain nucleotide sequence (13B Version 1) (SEQ ID NO: 30)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTGAAACAGGCACCAGGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGGGCCACCAACTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACAGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCCGCCTCCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCTGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTAG21R79 Heavy chain nucleotide sequence (13B Version 2) (SEQ ID NO: 31)ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTAATTATAATAGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAGTAG219R45 Heavy chain nucleotide sequence (13A Version 1) (SEQ ID NO: 32)ATGAAGCATCTGTGGTTTTTCCTGTTGCTCGTGGCGGCACCCAGATGGGTGTTGTCCCAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCAAGCGTAAAAGTATCGTGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGGTGCGGCAGGCTCCGGGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGCAGAACATCATATAAGGAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCTCGACTGCGTACATGGAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGCACCATCCATTATGATGACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGGTCACCGTCTCCAGCGCGTCGACGAAAGGTCCCTCGGTATTTCCCCTCGCCCCCTGCTCGAGGTCGACATCCGAATCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAGAGCCGGTAACGGTGTCGTGGAACTCGGGAGCGCTTACGTCCGGAGTCCACACATTTCCGGCGGTACTGCAATCCTCGGGACTGTATTCGTTGTCGTCAGTGGTGACTGTCCCGTCCTCCAATTTCGGGACTCAGACCTATACGTGCAACGTCGACCACAAACCCTCAAACACCAAGGTGGATAAGACAGTGGAGCGCAAGTGCTGCGTGGAGTGTCCCCCGTGTCCGGCACCCCCTGTCGCCGGACCCTCAGTCTTTTTGTTTCCGCCGAAGCCCAAAGATACACTCATGATCTCAAGAACGCCCGAGGTAACATGCGTGGTGGTCGATGTAAGCCACGAGGATCCAGAAGTACAATTCAATTGGTATGTAGACGGGGTCGAGGTCCATAACGCAAAGACGAAACCGAGGGAAGAGCAGTTCAATTCGACTTTCCGGGTGGTGTCGGTGCTTACAGTCGTACATCAGGACTGGTTGAACGGGAAGGAGTACAAGTGTAAAGTATCGAATAAGGGCCTTCCAGCGCCGATTGAAAAGACCATCTCCAAGACCAAAGGACAGCCACGAGAGCCGCAAGTCTATACGCTTCCTCCCAGCCGAGAAAAGATGACTAAAAACCAGGTATCGCTTACGTGTCTCGTCAAGGGTTTCTACCCTTCGGACATCGCGGTGGAATGGGAGAGCAATGGACAACCGGAAAACAACTACAAGACGACACCGCCTATGTTGAAAAGCGATGGATCGTTTTTCCTCTATTCGAAACTCACGGTCGATAAGTCACGGTGGCAGCAGGGGAATGTGTTCTCCTGTTCAGTGATGCACGAGGCGCTCCACAATCACTATACCCAGAAAAGCCTGTCACTTTCCCCGGGAAAATGA219R45 Heavy chain nucleotide sequence (13A Version 2) (SEQ ID NO: 33)ATGAAGCACCTCTGGTTCTTCCTGCTCCTCGTGGCTGCTCCTCGGTGGGTCCTCTCCCAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCTTCCGTCAAAGTCTCCTGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGGTGCGGCAGGCTCCTGGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGCAGAACCTCCTACAAGGAAAAATTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCTCCACAGCTTACATGGAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCACCATCCACTACGACGACAAGTACTACCCTCTGATGGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCCAGCGCTTCCACAAAAGGACCCTCCGTCTTTCCCCTCGCCCCCTGCTCCCGGTCCACATCCGAATCAACAGCTGCCCTCGGCTGCCTGGTCAAAGACTACTTCCCAGAGCCTGTCACAGTGTCCTGGAACTCCGGAGCTCTCACATCCGGAGTCCACACATTTCCTGCTGTGCTCCAATCCTCCGGACTGTATTCCCTCTCCTCCGTGGTGACAGTGCCTTCCTCCAATTTCGGGACACAGACCTATACATGCAACGTGGACCACAAACCCTCCAACACCAAAGTCGATAAGACAGTGGAGCGCAAGTGCTGCGTGGAGTGTCCCCCTTGTCCTGCTCCCCCTGTGGCTGGACCTTCCGTCTTTCTGTTTCCTCCTAAACCTAAAGACACCCTCATGATCTCCCGGACCCCCGAGGTCACATGCGTGGTCGTCGATGTGAGCCACGAGGACCCCGAAGTCCAATTTAATTGGTATGTGGACGGGGTGGAGGTCCATAACGCTAAGACCAAACCTAGGGAAGAGCAGTTCAATTCCACTTTCCGGGTGGTGTCCGTGCTGACCGTCGTTCATCAGGACTGGCTCAACGGGAAAGAATACAAATGCAAAGTCTCTAATAAGGGCCTCCCTGCTCCTATTGAAAAAACAATTTCCAAAACAAAAGGACAACCTCGGGAGCCTCAAGTCTACACACTGCCACCTTCCCGGGAAAAAATGACAAAAAATCAAGTCTCCCTCACATGTCTCGTCAAGGGATTCTACCCTTCCGACATTGCTGTGGAATGGGAATCCAATGGACAACCTGAAAACAACTACAAGACAACACCTCCTATGCTCAAAAGCGATGGGTCCTTTTTCCTCTATTCCAAACTCACAGTCGATAAGTCTCGGTGGCAGCAGGGGAATGTGTTCTCCTGTTCCGTGATGCACGAGGCTCTCCACAATCACTATACCCAGAAAAGCCTGTCCCTCTCCCCTGGAAAATGA Light chain nucleotide sequence (SEQ ID NO: 34)ATGGTGCTGCAGACCCAGGTGTTCATCTCCCTGCTGCTGTGGATCTCCGGCGCCTACGGCGACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGAGAGCGGGCCACCATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCATGAAGTGGTTCCAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCCAACCAGGGATCTGGCGTGCCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAAGAGGTGCCCTGGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCCGGAACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGTTAG21M18 Heavy chain variable region nucleotide sequence (SEQ ID NO: 35)CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCTTACTACATCCACTGGGTCAAGCAGGCCCCTGGGCAGGGCCTGGAATGGATCGGCTACATCTCCTCCTACAACGGCGCCACCAACTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCCTCT21R79 Heavy chain variable region nucleotide sequence (13B)(SEQ ID NO: 36)CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTGAAACAGGCACCAGGCCAGGGACTGGAATGGATCGGCTATATCGCCAACTACAACCGGGCCACCAACTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCCACCTCCACAGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCTCC21R79 Heavy chain variable region nucleotide sequence(13B Version 2) (SEQ ID NO: 37)CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTAATTATAATAGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT219R45 Heavy chain variable region nucleotide sequence(13A version 1) (SEQ ID NO: 38)CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCAAGCGTAAAAGTATCGTGTAAGGCCTCGGGGTACACGTTTACAAACTACTGGATGCATTGGGTGCGGCAGGCTCCGGGACAGGGGTTGGAATGGATGGGTGACATTAACCCCTCAAATGGCAGAACATCATATAAGGAAAAGTTCAAACGCCGCGTCACACTCTCCGTGGACAAGTCAAGCTCGACTGCGTACATGGAACTTTCGTCGCTGAGGTCGGAGGACACGGCAGTGTACTTTTGCACCATCCATTATGATGACAAGTATTACCCTCTGATGGATTATTGGGGTCAGGGTACGTTGGTCACCGTCTCC AGC219R45 Heavy chain variable region nucleotide sequence(13A Version 2) (SEQ ID NO: 39)CAAGTGCAGCTGGTCCAGAGCGGGGCTGAGGTGAAGAAACCCGGAGCTTCCGTCAAAGTCTCCTGTAAGGCTTCCGGATACACCTTTACCAACTATTGGATGCACTGGGTGCGGCAGGCTCCTGGACAAGGGCTGGAATGGATGGGAGACATCAATCCTTCCAATGGCAGAACCTCCTACAAGGAAAAATTCAAACGGCGGGTCACACTCTCCGTGGACAAGTCTAGCTCCACAGCTTACATGGAACTCTCCTCCCTGCGGTCCGAAGACACAGCTGTCTACTTCTGCACCATCCACTACGACGACAAGTACTACCCTCTGATGGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCC AGCLight chain variable region nucleotide sequence (SEQ ID NO: 40)GACATCGTGATGACCCAGTCCCCAGACTCCCTGGCTGTGTCTCTGGGAGAGCGGGCCACCATCTCTTGCAGAGCCTCCGAGTCCGTGGACAACTACGGCATCTCCTTCATGAAGTGGTTCCAGCAGAAGCCCGGCCAGCCCCCAAAGCTGCTGATCTACGCCGCCTCCAACCAGGGATCTGGCGTGCCCGACCGGTTCTCTGGATCCGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGTCCAAAGAGGTGCCCTGGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGHuman IgG1 Heavy chain constant region (SEQ ID NO: 41)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG2 Heavy chain constant region (SEQ ID NO: 42)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG3 Heavy chain constant region (SEQ ID NO: 43)ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgG4 Heavy chain constant region (SEQ ID NO: 44)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK FLAG peptide (SEQ ID NO: 45) DYKDDDDKParental 21R79 Heavy chain with signal sequence underlinedunmodified chain (SEQ ID NO: 46)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGOGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKParental 219R45 Heavy chain with signal sequence underlined(SEQ ID NO: 47)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKParental 21R79 Heavy chain without predicted signal sequence(SEQ ID NO: 48)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIANYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKParental 219R45 Heavy chain without signal sequence (SEQ ID NO: 49)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWMGDINPSNGRTSYKEKFKRRVTLSVDKSSSTAYMELSSLRSEDTAVYFCTIHYDDKYYPLMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKParental 21R79 Heavy chain variable region nucleotidesequence (SEQ ID NO: 50)CAAGTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGCATCGGTCAAAATCTCGTGTAAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGGTAAAGCAGGCGCCAGGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGCGCGACGAACTATAACCAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACATCGACGGCCTACATGGAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGCGCAAGAGATTACGACTATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAGTCTCCTCCParental 219R45 Heavy chain variable region nucleotidesequence (SEQ ID NO: 51)CAGGTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGCGTCGGTAAAGGTCAGCTGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGGTCAGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGACGGACTAGCTATAAGGAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCCTCAACGGCCTACATGGAGTTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTACTATCCACTATGACGATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTGGTAACCGTGAGC AGCParental 21R79 Heavy chain nucleotide sequence with signalsequence (SEQ ID NO: 52)ATGAAACACTTGTGGTTTTTCCTCTTGCTCGTGGCAGCTCCTCGGTGGGTACTTTCACAAGTGCAGCTCGTGCAGTCAGGGGCGGAGGTCAAGAAGCCGGGAGCATCGGTCAAAATCTCGTGTAAGGCCTCGGGGTACTCCTTTACTGCGTATTACATCCATTGGGTAAAGCAGGCGCCAGGGCAGGGATTGGAGTGGATTGGGTATATCGCCAATTACAATCGCGCGACGAACTATAACCAGAAATTCAAGGGAAGGGTGACCTTCACAACGGATACATCGACATCGACGGCCTACATGGAACTTCGCAGCCTGCGATCAGATGACACGGCGGTATACTATTGCGCAAGAGATTACGACTATGATGTGGGAATGGACTATTGGGGTCAAGGTACTCTGGTCACAGTCTCCTCCGCCAGCACCAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Parental 219R45 Heavy chain nucleotide sequence with signalsequence (SEQ ID NO: 53)ATGAAACACCTCTGGTTCTTTTTGCTCCTGGTGGCAGCTCCCCGATGGGTGCTTAGCCAGGTACAGCTCGTGCAATCGGGGGCAGAGGTCAAAAAGCCCGGTGCGTCGGTAAAGGTCAGCTGCAAAGCGTCAGGTTATACATTCACGAATTACTGGATGCATTGGGTCAGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGAGATATCAATCCGTCGAACGGACGGACTAGCTATAAGGAGAAGTTTAAGAGGCGCGTAACACTGTCGGTGGACAAATCGTCCTCAACGGCCTACATGGAGTTGTCATCCCTGCGGTCGGAAGATACGGCGGTCTACTTCTGTACTATCCACTATGACGATAAGTACTACCCGCTTATGGACTACTGGGGTCAGGGAACATTGGTAACCGTGAGCAGCGCGTCCACAAAGGGCCCTAGCGTCTTCCCTCTGGCTCCCTGCAGCAGGAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA Parental 21R79 and 219R45 light chain variable regionnucleotide sequence (SEQ ID NO: 54)GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACCATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTCCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCCGGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGGACATTCGGAGGAGGGACCAAGGTGGAAATCAAAParental 21R79 and 219R45 light chain nucleotide sequence(SEQ ID NO: 55)ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCGGAGCCTACGGGGACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACCATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTCCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCCGGGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGGACATTCGGAGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCCCCCTCCGTCTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAAAGCGGCACTGCCAGCGTGGTGTGCCTGCTGAATAACTTCTATCCCCGGGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAAAGCGGCAACTCCCAGGAGAGCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCCGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCAGCCCCGTCACAAAGAGCTTCAACAGGGGCGAGTGTTGA21R75 Heavy chain without predicted signal sequence (SEQ ID NO: 56)NQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R75 Heavy chain with predicted signal sequence (underlined)(SEQ ID NO: 57)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGOGLEWIGYIAGYKDATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R75 Heavy chain variable region (SEQ ID NO: 58)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYIAGYKDATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21R75 Heavy chain CDR2 (SEQ ID NO: 59) YIAGYKDATNYNQKFKG21R75 Heavy chain nucleotide sequence with signal sequence(13B Version 1) (SEQ ID NO: 60)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTACAACCAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCCCGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 21R75 Heavy chain nucleotide sequence with signal sequence(13B Version 1T) (SEQ ID NO: 77)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCTCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTACAACCAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCCCGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 21R75 Heavy chain nucleotide sequence with signal sequence(13B Version S1-2) (SEQ ID NO: 61)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAG21R83 Heavy chain without predicted signal sequence (SEQ ID NO: 62)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISNYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R83 Heavy chain with predicted signal sequence (underlined)(SEQ ID NO: 63)MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGOGLEWIGYISNYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK21R83 Heavy chain variable region (SEQ ID NO: 64)QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISNYNRATNYNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS21R83 Heavy chain CDR2 (SEQ ID NO: 65) YISNYNRATNYNQKFKG21R83 Heavy chain nucleotide sequence with signal sequenceunderlined (13B Version 1) (SEQ ID NO: 66)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCCCGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 21R83 Heavy chain nucleotide sequence with signal sequenceunderlined (13B Version 1T) (SEQ ID NO: 78)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCTCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTGCTCCAGATCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCTCCAACTTCGGCACCCAGACCTACACCTGTAACGTGGACCACAAGCCCTCCAACACCAAGGTGGACAAGACCGTGGAACGGAAGTGCTGCGTGGAATGCCCCCCTTGTCCTGCCCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCCATCGAAAAGACCATCTCTAAGACCAAGGGACAGCCCCGCGAGCCCCAGGTGTACACACTGCCTCCATCCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCATGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCGAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG 21R75 Heavy chain nucleotide sequence with signal sequenceunderlined (13B Version S1-2) (SEQ ID NO: 67)ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCCAGATGGGTGCTGTCCCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAG 21R75 Heavy chain variable region nucleotide sequence(13B Version 1) (SEQ ID NO: 68)CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTATATCGCCGGCTACAAGGACGCCACCAACTACAACCAGAAATTCAAGGGCAGAGTGACCTTCACCACCGACACCTCCACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCCTCT21R75 Heavy chain variable region nucleotide sequence(13B Version 2) (SEQ ID NO: 69)CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT21R83 Heavy chain variable region nucleotide sequence(13B Version 1) (SEQ ID NO: 70)CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGGCCTCCGGCTACTCCTTCACCGCCTACTACATCCACTGGGTCAAGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCTACATCTCCAACTACAACCGGGCCACCAATTACAACCAGAAATTCAAGGGCCGCGTGACCTTCACCACCGACACCTCTACCTCTACCGCCTACATGGAACTGCGGTCCCTGCGGAGCGACGACACCGCCGTGTACTACTGCGCCAGAGACTACGACTACGACGTGGGCATGGACTACTGGGGCCAGGGCACACTCGTGACCGTGTCTAGC21R75 Heavy chain variable region nucleotide sequence(13B Version 2) (SEQ ID NO: 71)CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT21R83 Heavy chain nucleotide sequence with signal sequenceunderlined (13B Version 2) (SEQ ID NO: 72)ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAGTAG 21R83 Heavy chain variable region nucleotide sequence(13B Version 2) (SEQ ID NO: 73)CAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCTCCAATTATAATAGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT21R75 Heavy chain nucleotide sequence with signal sequenceunderlined (13B Version 2) (SEQ ID NO: 74)ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCGCTGGATATAAAGATGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAGTAG21M18 Heavy chain nucleotide sequence (version 2) (SEQ ID NO: 75)ATGAAGCACCTATGGTTCTTTCTATTATTAGTGGCCGCTCCCCGTTGGGTGTTATCGCAGGTTCAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTGCATCCACTAAGGGACCATCCGTGTTCCCTTTGGCCCCTTGCTCTCGTTCGACCTCTGAATCGACTGCCGCTCTGGGATGCCTCGTGAAAGATTACTTCCCTGAGCCTGTGACCGTTTCCTGGAACTCGGGCGCCCTAACCTCTGGCGTGCACACATTCCCTGCCGTGCTACAGTCTTCTGGCCTATACTCTTTATCTTCGGTTGTTACCGTACCTTCTTCTAACTTCGGAACCCAAACTTACACCTGTAACGTAGACCACAAGCCTTCGAACACCAAGGTGGACAAGACTGTTGAGCGAAAGTGCTGCGTTGAGTGCCCTCCATGTCCTGCACCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCAAAACCTAAGGACACTCTAATGATCTCTCGGACTCCTGAGGTGACTTGCGTGGTTGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGAGTCGAGGTGCACAATGCAAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTTTCTGTGTTGACCGTTGTGCACCAAGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCAGCCGGGAAGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGGAGGGCTTCTACCCTTCCGACATCGCCGTTGAGTGGGAGTCTAACGGACAGCCGGAGAACAACTACAAGACTACGCCTCCAATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCGAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCATGCTCCGTAATGCACGAAGCCTTGCACAATCACTACACTCAAAAGTCCCTATCCTTATCTCCTGGCAAGTAG21M18 Heavy chain variable region (version 2) (SEQ ID NO: 76)CAGCTAGTTCAGTCTGGAGCGGAAGTTAAGAAACCTGGAGCATCCGTGAAAATAAGTTGCAAGGCATCCGGTTACTCGTTCACCGCATACTATATCCACTGGGTTAAACAGGCACCAGGACAGGGACTTGAATGGATCGGATATATCTCCTCTTATAATGGAGCTACAAACTATAACCAAAAATTCAAAGGACGCGTGACTTTCACAACTGACACCTCAACCTCGACAGCATACATGGAATTACGGTCCCTACGGTCTGACGACACTGCCGTTTACTATTGCGCTAGAGATTATGATTATGATGTTGGAATGGACTATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCTAnti-DLL4 heavy chain CDR2 consensus sequence (SEQ ID NO: 80):YIX₁X₂YX₃X₄ATNYNQKFKG,where X₁ is serine or alanine, X₂ is serine, asparagine, orglycine, X₃ is asparagine or lysine, and X₄ is lysine, arginine,or aspartic acid

1-89. (canceled)
 90. An isolated polynucleotide encoding a modifiedimmunoglobulin molecule, wherein the modified immunoglobulin moleculecomprises: (a) a first antigen-binding site that specifically bindshuman VEGF; and (b) a second antigen-binding site that specificallybinds human DLL4; wherein the first antigen-binding site comprises aheavy chain CDR1 comprising NYWMH (SEQ ID NO:17), a heavy chain CDR2comprising DINPSNGRTSYKEKFKR (SEQ ID NO:18), and a heavy chain CDR3comprising HYDDKYYPLMDY (SEQ ID NO:19); wherein the secondantigen-binding site comprises a heavy chain CDR1 comprising TAYYIH (SEQID NO:13) or AYYIH (SEQ ID NO:79), a heavy chain CDR2 comprisingYIANYNRATNYNQKFKG (SEQ ID NO:14), YISSYNGATNYNQKFKG (SEQ ID NO:15),YIAGYKDATNYNQKFKG (SEQ ID NO:59), or YISNYNRATNYNQKFKG (SEQ ID NO:65),and a heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); andwherein both the first and second-antigen binding sites comprise a lightchain having at least 95% sequence identity to SEQ ID NO:4 that lackssignal sequence MVLQTQVFISLLLWISGAYG (amino acid residues 1-20 of SEQ IDNO:4).
 91. The polynucleotide of claim 90, wherein both the first andsecond antigen-binding sites comprise a light chain comprising SEQ IDNO:4 that lacks signal sequence MVLQTQVFISLLLWISGAYG (amino acidresidues 1-20 of SEQ ID NO:4).
 92. The polynucleotide of claim 90,wherein the light chain further comprises a light chain CDR1 comprisingRASESVDNYGISFMK (SEQ ID NO:20), a light chain CDR2 comprising AASNQGS(SEQ ID NO:21), and light chain CDR3 comprising QQSKEVPWTFGG (SEQ IDNO:22).
 93. The polynucleotide of claim 90, wherein the heavy chainvariable region of the second antigen-binding site comprises (i) a heavychain CDR1 comprising TAYYIH (SEQ ID NO:13), (ii) a heavy chain CDR2comprising YISNYNRATNYNQKFKG (SEQ ID NO:65), and (iii) a heavy chainCDR3 comprising RDYDYDVGMDY (SEQ ID NO:16).
 94. An isolatedpolynucleotide encoding a modified immunoglobulin molecule, wherein themodified immunoglobulin molecule comprises: (a) a first antigen-bindingsite that specifically binds human VEGF; and (b) a secondantigen-binding site that specifically binds human DLL4; wherein thefirst antigen-binding site comprises a heavy chain CDR1 comprising NYWMH(SEQ ID NO:17), a heavy chain CDR2 comprising DINPSNGRTSYKEKFKR (SEQ IDNO:18), and a heavy chain CDR3 comprising HYDDKYYPLMDY (SEQ ID NO:19);wherein the second antigen-binding site comprises a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13) or AYYIH (SEQ ID NO:79), a heavy chainCDR2 comprising YIX₁X₂YX₃X₄ATNYNQKFKG (SEQ ID NO:80), wherein X₁ isserine or alanine, X₂ is serine, asparagine, or glycine, X₃ isasparagine or lysine, and X₄ is glycine, arginine, or aspartic acid, anda heavy chain CDR3 comprising RDYDYDVGMDY (SEQ ID NO:16); and whereinboth the first and second-antigen binding sites comprise a light chainhaving at least 95% sequence identity to SEQ ID NO:4 that lacks signalsequence MVLQTQVFISLLLWISGAYG (amino acid residues 1-20 of SEQ ID NO:4).95. The polynucleotide of claim 94, wherein both the first and secondantigen-binding sites comprise a light chain comprising SEQ ID NO:4 thatlacks signal sequence MVLQTQVFISLLLWISGAYG (amino acid residues 1-20 ofSEQ ID NO:4).
 96. The polynucleotide of claim 94, wherein the lightchain further comprises a light chain CDR1 comprising RASESVDNYGISFMK(SEQ ID NO:20), a light chain CDR2 comprising AASNQGS (SEQ ID NO:21),and light chain CDR3 comprising QQSKEVPWTFGG (SEQ ID NO:22).
 97. Thepolynucleotide of claim 94, wherein the heavy chain variable region ofthe second antigen-binding site comprises (i) a heavy chain CDR1comprising TAYYIH (SEQ ID NO:13), (ii) a heavy chain CDR2 comprisingYISNYNRATNYNQKFKG (SEQ ID NO:65), and (iii) a heavy chain CDR3comprising RDYDYDVGMDY (SEQ ID NO:16).
 98. A composition comprising thepolynucleotide of claim
 90. 99. A composition comprising thepolynucleotide of claim
 94. 100. A vector comprising the polynucleotideof claim
 90. 101. A vector comprising the polynucleotide of claim 94.102. An isolated cell comprising the vector of claim
 90. 103. Anisolated cell comprising the vector of claim
 94. 104. An isolatedpolynucleotide encoding an antibody that specifically binds VEGF,wherein the antibody comprises: (a) a heavy chain having the sequence ofSEQ ID NO:49 or SEQ ID NO:7; and (b) a light chain having the sequenceof SEQ ID NO:4 that lacks signal sequence MVLQTQVFISLLLWISGAYG (aminoacid residues 1-20 of SEQ ID NO:4).
 105. The polynucleotide of claim104, wherein the antibody comprises: (a) a heavy chain variable regionhaving the sequence of SEQ ID NO:11; and (b) a light chain variableregion having the sequence of SEQ ID NO:12.
 106. A compositioncomprising the polynucleotide of claim
 104. 107. A compositioncomprising the polynucleotide of claim
 105. 108. A vector comprising thepolynucleotide of claim
 104. 109. An isolated cell comprising the vectorof claim 104.